ES2368154T3 - PROCEDURES FOR ADAPTATION OF THE IMMUNE RESPONSE TO AN ANTIGEN OR IMMUNOGEN. - Google Patents

Abstract

A primary preparation for intramuscular administration comprising a nucleic acid encoding an antigen: and a secondary preparation for intranasal administration comprising the antigen encapsulated in liposomes, to elicit an immune response in an animal in which the administration of the secondary preparation produces a immune response comprising: (1) an increase in the serum level of the antigen specific IgA compared to the serum level of the antigen specific IgA prior to administration of the secondary preparation and, (2) an increase in Comparison with an untreated animal in the proportion of antigen-specific CD8-IFNγ + cytotoxic T lymphocytes in both the lung and spleen.

Description

Procedures for adapting the immune response to an antigen or immunogen

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. Provisional Application No. 60 / 534,923, filed on January 7, 2004.

GOVERNMENT FINANCING

The invention described in this document was supported, in whole or in part, by Grant R31 / CCR922413-01; R31 / CCR924378-01 of the Centers for Disease Control and Prevention. The United States government has certain rights over the invention.

BACKGROUND OF THE INVENTION

The development of effective preventive and therapeutic vaccines against a variety of agents (such as infectious microorganisms) is an important goal for disease control worldwide. In many cases, the most effective vaccine specifically targets the most common portal of entry for microorganisms, mucous surfaces of the body, including the nasal passages, the lungs, the reproductive tracts and the intestine.

Protective immunity against specific pathogens is achieved by humoral, cellular and mucosal immune responses. The humoral or antibody responses are important in the neutralization of pathogens and can be very effective in some infectious diseases. Antibodies circulate in the blood, and provide a measure of total humoral immune responses. Cell-mediated immunity, specifically the involvement of cytotoxic T lymphocytes (CTL) is necessary for protective immunity against many intracellular pathogens and cancer. Immunity located on the mucous surfaces of the nasal passages, lungs, reproductive tracts and intestines is also crucial to produce effective immune responses against pathogens that access the body through these sites. The hallmark of effective mucosal immune responses is the local production of secretory IgA antibody on the mucous surfaces. IgA also circulates in the blood and its levels in it provide an estimate of the total mucosal immune responses. Mucosal IgG is also important in the neutralization of certain pathogens. Repeated immunization may result in improved immune responses and may confer increased protection. A primary immunization establishes a base level of immunity and can lead to the development of T and B memory lymphocytes. A secondary immunization at a later time can mobilize these memory lymphocytes and lead to greater and more specific immune responses.

Most of the immune responses are regulated by T lymphocytes, which initiate and shape the nature of the response. As immune responses mature, CD4 + T lymphocytes can polarize toward immune responses of collaborating T type 1 (Th1) or T helper type 2 (Th2). The hallmark of the Th1 and Th2 type responses is the predominant pattern of the cytokines that are present. Th1 responses are characterized by high levels of IFN-γ and low levels of IL-4 and IL-10, while Th2 responses are characterized by low levels of IFN-γ and high levels of IL-4 and IL- 10. These cytokines play an important role in determining the functional capabilities of T lymphocytes. Th2 type responses lead to the preferred production of antibodies of the IgG1 subclass, with little or no CTL generation. Th1-type responses lead to the preferred production of antibodies of the IgG2a subclass and the induction of CTL that can effectively kill cells infected with viruses or other organisms.

Table A below summarizes the immunological characteristics of the polarized immune responses of Th1 and Th2. Th1 polarized responses are typically generated during virus infections

or bacteria In contrast, polarized Th2 responses are often observed in infection is parasitic, in allergic responses and by conventional intramuscularly administered protein vaccines based on alum used in humans. Genetics can also determine the type of immune response generated. For example, Th1 responses predominate in the mouse C57BL / 6 strain, while Th2 responses predominate in the mouse Balb / c strain. The immune responses may also contain both Th1 and Th2 components, providing protection by both humoral and cell-mediated immune response arms. Direct determination of the frequencies of the cytokine-producing cells is performed through the use of ELISPOT (Enzyme-linked Immunoabsorbent Point assays) or by immunofluorescent staining to reveal intracellular cytokine production. The proportions of serum IgG1: IgG2a are also a widely accepted and followed criterion for determining the types of collaborating T (Table A). A proportion of IgG1 with respect to IgG2a to obtain a balanced Th1 and Th2 response must be between 0.5 and 2.0.

Table A: Characteristics of Th1 and Th2 responses of polarized T lymphocytes

Immune responses

Th1 type immune responses Th2 type immune responses

Humoral immunity

Serum IgG1 / IgG2a <0.5 Serum IgG1 / IgG2a> 2.0

T lymphocyte cytokine secretion

↑ IFN-γ, ↓ IL-10 and IL-4 ↑ IL-10 and IL-4, ↓ IFN-γ,

CTL

High Low or absent

Prototype mouse strains

C57BL / 6 Balb / c

As used in this document, &quot; response of T collaborators of type 1 &quot; and "Th1 response" They are used interchangeably to refer to a range of host animal responses that include one or more, usually all, of the characteristics listed in the central column of Table A above. These characteristics include a ratio of IgG1: IgG2a of no more than 0.5; increased IFN-γ secretion (and other Th1 cytokines) by helper T lymphocytes 1 and reduced IL-10 and IL-4 secretion (and other Th2 cytokines) by helper T lymphocytes 2; and a high activity of CTL.

Similarly, as used in this document, "the response of T collaborators of type 2" and "Th2 response" They are used interchangeably to refer to a range of host animal responses that include one or more, usually all, of the characteristics listed in the right column of Table A above. These features include a ratio of IgG1: IgG2a of no more than 2.0; secretion of reduced IFN-γ (and other Th1 cytokines) by helper T lymphocytes 1 and increased IL-10 and IL-4 secretion (and other Th2 cytokines) by helper T lymphocytes 2; and a low or no CTL activity.

The administration of the vaccine takes a variety of forms, depending on the agent to be administered and the route of administration. Vaccine delivery systems are often designed to administer vaccines to specific areas of the body. In the gastrointestinal tract it is important that the vaccine is not degraded or eliminated before it has had the opportunity to exert a localized effect or to pass into the bloodstream or to interact with lymphoid tissue in the local environment. In the nasopharyngeal tract, it is important that the vaccine remains in close proximity to the absorbed cells; It is also important that the antigens remain in contact with the lymphoid cells before the antigen is washed through the nasal tract or ingested. In addition, it is important that the antigen, vehicle and immune stimulator co-administer in a single complex.

Traditionally, immunization has been performed by administering inactivated whole organisms or cells, extracts of microorganisms or cells, or components isolated therefrom, such as protein or peptide antigens. Immunization has been performed by oral, intranasal (IN) or intramuscular (IM) administration of live attenuated organisms. Typically, such compositions and the presence of preservatives, adjuvants and other excipients are injected into humans and animals intramuscularly or subcutaneously to induce protective immunity in normal pediatric or common-use adult vaccines. Parenteral immunization is rarely able to elicit an effective mucosal immune response that results in the production of antibodies, particularly the production of immunoglobulin A (IgA), which is important as a first defense barrier for the invasion of microorganisms.

Currently, only live attenuated viruses or bacteria are capable of inducing a protective immune response in humans or animals when administered orally or intranasally. To date, very few vaccines have been developed that can be administered orally, intranasally or intravaginally, and these vaccines are invariably living organisms, such as oral poliovirus vaccines and an oral typhoid fever vaccine. commonly used salmonella. For example, the cold-attenuated live flu virus prepared by cold adaptation (FluMist ™) can be used to vaccinate humans against the flu by intranasal administration. There has been concern about the potential risks associated with the use of vector or live virus-based vaccines due to the natural tendency of genetic mutation, as well as efficacy and safety problems when applied in immunosuppressed patients.

Typically, commonly used vaccines are prepared from cell extracts or produced by a recombinant production methodology; These vaccines are normally composed of proteins, microbial toxoids, inactivated whole viruses or polysaccharides of microbial origin. These types of materials are usually effective in inducing protective immune responses against diseases when administered by injection. However, such materials are poorly immunogenic or non-immunogenic when administered through oral or intranasal administration, even if the intestinal and nasopharyngeal tract of humans and most animals are rich in cells and tissues capable of induction of the immune response to non-host antigens. One of the main failures of vaccines administered orally or intranasally is that the antigens are poorly absorbed and unstable during the passage. The proteins and peptides that are administered orally are invariably degraded in the GI tract by the action of proteases and other hydrolytic enzymes, and stomach acids. Thus, vaccines composed of subcellular immunogens are ineffective when administered orally or intranasally. Therefore, compositions that can be used repeatedly as antigen-protective vehicles, as well as effective adjuvants that can induce effective immune responses after oral or intranasal administration will provide a more useful and convenient means of vaccinating animals and humans.

The Mucous Associated Lymphoid Tissue (MALT) is a network system consisting of the gastrointestinal tract (lymphoid tissue associated with the intestine, GALT), mammary glands, reproductive tract and respiratory tract (lymphoid tissue associated with the bronchial and nasopharynx, BALT and NALT). One of the unique features of the common mucosal immune system is that immune induction at a mucosal site often results in immune responses at distal mucosal sites. This is due to the migration of lymphocytes through the high endothelial venules. However, clear differences have been observed after the administration of vaccines to the different mucosal surfaces and that the compartmentalization of responses can occur. In addition, some mucosal sites induce better responses at distal sites and not all distal sites respond in the same way.

Intranasal immunization is an effective route for the exposure of mucosal immunity to various pathogens, soluble proteins and antigens administered by microparticles. Intranasal immunization has the added advantage of inducing both mucosal immune responses both locally and distally. For example, intranasal vaccination can obtain immunity from the genito-urinary tract, in which direct immunization is often hindered by the renewal of epithelial cells and hormonal influences. (Csencsits KL et al. 1999. JI. 1382-1389). Intranasal vaccination can induce strong specific immune responses of antigens through NALT, or through deep lung immunization. NALT, the main mucosal inducing site for the upper respiratory tract, is important for the development of mucosal immunity locally and distal to the antigen immunized intranasally. Mucosal IgA and IgG responses and high CTL responses to viral peptides have been observed after exposure to NALT. The lung is an immune-sensitive organ that can locally produce IgA, IgG, IgE and cell-mediated immune responses (Liu M et al. 1982. J Immunology. 129 (6): 26532661) (Bice DE, et al. , 1980. IntArch Allergy Appl Immunol. 63 (4): 438-45). Protective immune responses have been demonstrated in the lung after influenza virus infection or vaccination with the flu vaccine, while long-term antibody production after lung immunization and exposure have been related to tissues. associated to the lung (Bice DE, et al 1993. Am J Respir Cell Mol Biol. 8 (6): 662-667).

The Mucous Associated Lymphoid Tissue (MALT) is a network system consisting of the gastrointestinal tract (lymphoid tissue associated with the intestine, GALT), mammary glands, reproductive tract and respiratory tract (lymphoid tissue associated with the bronchial and nasopharynx, BALT and NALT). One of the unique features of the common mucosal immune system is that immune induction at a mucosal site often results in immune responses at distal mucosal sites. This is due to the migration of lymphocytes through the high endothelial venules. However, clear differences have been observed after the administration of vaccines to the different mucosal surfaces and that the compartmentalization of responses can occur. In addition, some mucosal sites induce better responses at distal sites and not all distal sites respond in the same way.

Intranasal immunization is an effective route for the exposure of mucosal immunity to various pathogens, soluble proteins and antigens administered by microparticles. Intranasal immunization has the added advantage of inducing both mucosal immune responses both locally and distally. For example, intranasal vaccination can obtain immunity from the genito-urinary tract, in which direct immunization is often hindered by the renewal of epithelial cells and hormonal influences. (Csencsits KL et al. 1999. JI. 1382-1389). Intranasal vaccination can induce strong specific immune responses of antigens through NALT, or through deep lung immunization. NALT, the main mucosal inducing site for the upper respiratory tract, is important for the development of mucosal immunity locally and distal to the antigen immunized intranasally. Mucosal IgA and IgG responses and high CTL responses to viral peptides have been observed after exposure to NALT. The lung is an immune-sensitive organ that can locally produce IgA, IgG, IgE and cell-mediated immune responses (Liu M et al. 1982. J Immunology. 129 (6): 26532661) (Bice DE, et al. , 1980. IntArch Allergy Appl Immunol. 63 (4): 438-45). Protective immune responses have been demonstrated in the lung after influenza virus infection or vaccination with the flu vaccine, while long-term antibody production after lung immunization and exposure have been related to tissues. associated to the lung (Bice DE, et al 1993. Am J Respir Cell Mol Biol. 8 (6): 662-667). The optimal immunity of the upper respiratory tract is induced by the combined inoculation of the upper and lower respiratory tract (Thompson AH. Vaccine 17: 1404-15).

Immunization with DNA is a method for generating protective immunity against infectious diseases (Liu et al., Ann. N.Y. Acad. Sci. 772, 1995). Unlike vaccines of subunits based on proteins or peptides, immunization with DNA provides protective immunity through the expression of foreign proteins by host cells, thus allowing the presentation of the antigen to the immune system in a more similar way. which occurs during infection by intracellular viruses or pathogens (Pardoll and Beckerieg, Immunity 3: 165, 1995; McDonnell and Askari, N. Engl. J. Med. 334: 42, 1996). Although considerable interest in this technique has been generated, successful immunity has been more consistently induced by immunization with DNA for viral diseases (Manickan et al., J. Immunol. 155: 259, 1995). The results have been more variable with pathogenic non-viral pathogens that may reflect differences in the nature of the pathogens, in the chosen immunizing antigens and in the immunizing pathways (Sedegah et al., Proc. Natl. Acad. Sci. USA 91 : 9866, 1994; Barry et al., Nature 377: 632, 1995; Xu and Liew, Vaccine 12: 1534, 1994). Further development of DNA vaccination will depend on the elucidation of the underlying immune mechanisms and extension of their application to other infectious diseases for which current vaccine development strategies have failed.

DNA vaccines were well tolerated in humans according to several recent clinical trials, but there is an urgent need to achieve better immunizing potency. Therefore, several second-generation DNA vaccines using various systems and devices are under development. In addition, a new approach to immunization, called mixed modality vaccination or main vaccine, is being evaluated. It is an initial vaccination that uses one type of vaccine, followed by a booster that uses a different type of vaccine. For example, promising preclinical results have been obtained by first immunizing with DNA and then by boosting with a vaccine or an adenovirus vector encoding the same antigen,

or with a recombinant protein of the same antigen as the encoded DNA vaccine. WO-A00 / 11140 describes a method for eliciting an immune response in a mammal by administering a primary composition comprising a DNA sequence encoding an antigen followed by intranasal administration of a secondary composition comprising the DNA encoding the antigen. However, there are doubts about the vaccine and the adenovirus that is used as reinforcement. This is because the pre-existing antibody against the viral vector could block the immune response, these recombinant viruses usually cannot be used several times and there is also a safety problem especially for immunosuppressed patients. In addition, pure proteins generally used as reinforcement of parenteral immunizing pathways may not induce a mucosal immune response or improve the cell-mediated immune response.

Mucosal protective immune responses are crucial in the fight against diseases caused by many naturally occurring infectious organisms and microorganisms that can be used for bioterrorist purposes. Cell-mediated immune responses, including CTL, are also important in the control of intracellular pathogens, both systemically and in mucosal sites. Traditional intramuscularly administered protein vaccines, which use alum as an adjuvant, generate a typical Th2 response with mainly IgG1 antibody responses, but do not stimulate CTL responses or local immunity at mucosal sites. Therefore, there is a need for effective vaccines that are capable of producing a wide range of desired immune responses to protect the animal from disease.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a primary preparation for intramuscular administration comprising a nucleic acid encoding an antigen: and a secondary preparation for intranasal administration comprising the antigen encapsulated in liposomes, to elicit an immune response in an animal in the that the administration of the secondary preparation produces an immune response comprising:

(one)

an increase in the serum level of the antigen specific IgA compared with the serum level of the antigen specific IgA prior to administration of the secondary preparation and,

(2)

an increase compared to an untreated animal in the proportion of antigen-specific CD8-IFNγ + cytotoxic T lymphocytes in both the lung and spleen.

Preferred aspects of the composition are detailed in claims 2 to 10 and claim 18.

In another aspect, the invention relates to a kit for eliciting an immune response in a host animal comprising:

a) a first immunizing component for intramuscular administration comprising a nucleic acid encoding an antigen; b) a second immunizing component for intranasal administration comprising the antigen encapsulated in liposomes; c) an instruction for a user to administer to the animal the first immunizing component followed by administration of the second immunizing component to elicit an immune response in the animal. Preferred aspects of the kit are detailed in claims 12 to 17 and 19.

A method is also described for eliciting a desired immune response in an animal, which comprises administering to the animal a primary preparation comprising a nucleic acid sequence encoding an antigen and a secondary preparation comprising the antigen and liposomes (antigen-liposome preparation) ( heterologous immunization), thereby causing the desired immune response in the animal).

The desired immune response is against a pathogen and the antigen is a pathogen specific antigen.

The primary preparation is administered by a route selected from: subcutaneous route, intramuscular route, route

intradermally and mucosally, and the secondary preparation is administered by a route selected from:

subcutaneously, intramuscularly, intradermally and mucosa. A procedure is also described in which the primary preparation for heterologous immunization is administered intramuscularly to said animal, and wherein said secondary preparation is administered via intranasal to said animal.

The secondary preparation comprises a pathogen specific protein antigen encapsulated in liposomes. The liposomes in the secondary preparation have an average size of approximately 0.5-5 µm. Each of the primary preparation and secondary preparation, when administered alone in the animal, is

enough to perform the desired level of immune response. The immune response confers immunity against the pathogen. The priming comprises one or two administrations separated by approximately 3-6 weeks, and in which said

Secondary preparation is administered approximately 3 to 10 weeks after the last priming.

Priming comprises one or two administrations separated by 3 days, wherein said secondary preparation is administered 3 weeks after the last priming. The liposomes in said antigen-liposome preparation have an average size of approximately 0.5-5

µm. The secondary booster dose is administered intranasally to said animal to induce an IgA response. The IgA response includes systemic and mucosal response. A procedure is also described to cause an immune response tended to a Th2 response in a

animal. The procedure comprises administering a primary preparation and a secondary preparation, each

of which it comprises the same antigen and liposomes (homologous immunization). The host animal is a mammal, such as a human being or a non-human animal, including an animal of domestic cattle (cows, pigs, horses or sheep, etc.), a pet (dog or cat, etc.), or an animal of small experimentation (mouse or rat, etc.).

The pathogen is a virus, such as the Hepatitis B virus (HBV). Optionally, the pathogen is one of the Chlamydia strains. Optionally, the pathogen is a bacterial strain, such as Bacillus anthracis. Optionally, the pathogen specific antigen is a protective antigen (PA), such as the PA antigen of

Bacillus anthracis.

Optionally, the liposome trapped immunogen is a peptide that activates T lymphocytes or a virus particle whole inactivated. Optionally, the pathogen specific antigen is a viral surface antigen, such as HBsAg. Optionally, the pathogen specific antigen is a peptide. Optionally, the pathogen specific antigen is a type of inactivated virus or bacteria. Optionally, the pathogen-specific antigen (peptide, protein or inactivated virus) is encapsulated in

liposomes

Optionally, the pathogen-specific antigen (peptide, protein or inactivated virus / bacteria) is mixed with empty liposomes Optionally, the antigen-liposome preparation is freshly prepared. Optionally, the antigen-liposome preparation is lyophilized before use. Optionally, the method further comprises administering one or more secondary preparations of the

said antigen-liposome preparation itself (homologous immunization), in which each administration is separated by about 2-16 weeks, preferably about 3-6 weeks.

Optionally, all preparations have the same amount of said pathogen specific antigen. 5 Optionally, different amounts of primer and reinforcement are administered to said animal through the same route.

Optionally, different amounts of primary and secondary preparations are administered to said animal through separate routes.

Optionally, the secondary preparation or preparations and the initial priming are administered to said animal through the same route.

Optionally, the secondary preparation and initial priming are administered to said animal through separate routes.

Optionally, the method further comprises measuring humoral and / or cellular immune responses to said pathogen specific antigen.

20 Optionally, the humoral immune response includes titers of total antigen-specific antibodies (Ig) in serum or mucosal surfaces; titers of anti-HBsAg specific antibodies in serum or mucous surfaces; titres of isotypes and / or subtypes of antigen specific antibodies, including IgG, IgA, IgG1 and IgG2a; proportion of IgG1 and IgG2a.

Optionally, the cellular immune response includes the appearance of CD8 + IFN-γ + cytotoxic T lymphocytes (CTL); enhanced CTL destruction activity; secretion of cytokines characteristic of the Th1 response, including IFN-γ; cytokine secretion characteristic of the Th2 response, including IL-10 and IL-4; and polarization of the helper T lymphocyte profile (eg, Th1 response versus Th2 response).

Optionally, humoral and / or cellular immune responses are generally measured from samples obtained from said host animals approximately 2, 4, 6 or 8 weeks after the last boost. However, responses can be measured for many months after the last reinforcement.

Optionally, the immune response includes responses of IgA, IgG and T lymphocytes.

Optionally, the IgA response includes systemic and / or mucosal IgA response or responses.

Optionally, the IgG response includes systemic and / or mucosal IgG response or responses.

An immunogenic composition comprising two immunizing components is also described, in which the first immunizing component comprises a nucleic acid encoding an antigen, and the second immunizing component comprises the antigen and the liposome. Optionally, the desired immune response is against a pathogen and the antigen is a pathogen specific antigen.

45 BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the liposome size distribution profile of an average size of 4 µm in diameter. The x axis shows the size in µm and the y axis shows the number of particles.

Figure 2 is representative of the degree of serum variation of the specific Ig titers of HBsAg observed between individual CD1 (upper panel) and Balb / c (lower panel) mice in an experiment. The responses are shown from mice that received one (closed circles) or two (closed triangles) homologous immunizations with HBsAg IN liposomes, HBsAg IM liposomes, or the commercially available GSK vaccine for HBsAg IM. Terminal titers are defined as a serum titre> 2 x that was observed for untreated mice. The horizontal lines represent the average response (n = 5 mice per group).

Figure 3 illustrates the kinetics of HBsAg specific serum Ig levels in CD1 mice after intranasal immunization with HBsAg liposomes. The data is shown as the OD at 450 nm in the HBsAg-specific Ig ELISA after serial dilution of serum samples several times after primary immunization (closed symbols) and two weeks after secondary immunization (closed symbols with cross hatches, +). Values are averages from serum combinations (n = 5 mice per group). The responses to the GSK HBsAg vaccine are shown for comparison.

Figure 4 illustrates the dose response of HBsAg liposomes in CD1 mice immunized intranasally at week 0 (closed symbols) or at weeks 0 and 6 (closed symbols with cross hatching, +). The responses to the GSK HBsAg vaccine are shown for comparison. HBsAg specific serum Ig levels were measured in combinations of serum samples taken 8 weeks after the first immunization (n = 5 mice per group).

Figure 5 shows the effect of the route of administration and the delivery of a secondary immunization on a serum antibody specific for total HBsAg in CD1 mice administered with liposomes of HBsAg (15 µg / dose). The closed symbols show responses in mice (n = 5) that received a single primary immunization. Closed symbols with cross hatching (+) show responses in mice (n = 5) that received identical homologous primary and secondary booster immunizations. The responses to the GSK HBsAg vaccine are shown for comparison.

Figure 6 shows the effect of the route of administration and the delivery of a secondary immunization on a serum antibody specific for total HBsAg in Balb / c mice administered with liposomes of HBsAg (15 µg / dose). The closed symbols show responses in mice (n = 5) that received a single primary immunization. Closed symbols with cross hatching (+) show responses in mice (n = 5) that received identical homologous primary and secondary booster immunizations. The responses to the GSK HBsAg vaccine are shown for comparison.

Figure 7 shows a comparison of HBsAg-specific serum Ig responses in CD1 mice immunized intranasally with fresh (square) or lyophilized HBsAg liposomes (triangles), or immunized intramuscularly with the commercially available GSK vaccine. (circles) Closed symbols show responses in mice (n = 5) that received a single primary immunization. Closed symbols with cross hatching (+) show responses in mice (n = 5) that received homologous primary and secondary booster immunizations. Total serum antibody responses of total HBsAg were measured in serum 8 weeks after primary immunization.

Figure 8 shows a comparison of HBsAg-specific serum Ig responses in CD1 mice immunized intramuscularly with fresh (square) or lyophilized HBsAg liposomes (triangles), or immunized intramuscularly with the commercially available GSK vaccine. (circles) Closed symbols show responses in mice (n = 5) that received a single primary immunization. Closed symbols with cross hatching (+) show responses in mice (n = 5) that received homologous primary and secondary booster immunizations. Total serum antibody responses of total HBsAg were measured in serum 8 weeks after primary immunization.

Figure 9 shows the effect of liposome size on HBsAg-specific serum IgG responses in Balb / c mice (6 weeks after primary immunization and 2 weeks after booster). Liposomes containing HBsAg (15 µm) had a size of 4 µm, 1 µm and 0.2 µm and were administered intranasally to Balb / c mice (n = 5) in a single size,

or in the form of an equal mixture of the three sizes. A quantitative ELISA for HBsAg specific IgG was used to determine serum IgG levels in combined serum samples (n = 5 mice per group). The vertical bar in the mixed group shows the predicted value if the antibody responses are additive.

Figure 10 shows the IgG1: IgG2a ratios of HBsAg specific antibodies in the serum of CD1 mice immunized by different protocols. The gray bars are the IgG1: IgG2a ratios of mice that received an immunization at week 0. The black bars are the IgG1: IgG2a ratios of mice that received 2 homologous immunizations at weeks 0 and 6. The specific quantitative ELISA assays of HBsAg were used to determine the level of IgG1 and specific IgG2a of HBsAg in the serum of a serum combination (equal amounts of serum from 4 or 5 individual mice per group) 8 weeks after primary immunization. Two vertical lines at 0.5 and 2.0 at each panel demarcate three different patterns of antibody responses. The proportion of 0.5 or less indicates a response with a tendency to Th1. A proportion of 2.0 or greater indicates a response with a tendency to Th2. The proportions between 0.5 and 2.0 indicate a mixed or balanced response.

Figure 11 shows the proportions of IgG1: IgG2a of HBsAg specific antibodies in the serum of CD1 mice immunized by different protocols. The gray bars are the IgG1: IgG2a ratios of mice that received an immunization at week 0. The black bars are the IgG1: IgG2a ratios of mice that received 2 homologous immunizations at weeks 0 and 6. The specific quantitative ELISA assays of HBsAg were used to determine the level of IgG1 and specific IgG2a of HBsAg in the serum of a serum combination (equal amounts of serum from 4 or 5 mice per treatment group) 8 weeks after primary immunization. Two vertical lines at 0.5 and 2.0 at each panel demarcate three different patterns of antibody responses. The proportion of 0.5 or less indicates a response with a tendency to Th1. A proportion of 2.0 or greater indicates a response with a tendency to Th2. The proportions between 0.5 and 2.0 indicate a mixed or balanced response.

Figure 12 shows that HBsAg DNA is a weak immunogen for antibody responses in CD1 (square) and Balb / c (triangles) mice. Responses to the GSK vaccine in CD1 mice (circles filled with cross shading, ±) and in Balb / c mice (open circles with cross shading, +) are shown for comparative purposes. Closed symbols show responses in mice (n = 5) that received a single primary immunization. Cross-hatched symbols (+) show responses in mice (n = 5) that received homologous primary and secondary booster immunizations. Total serum antibody responses of total HBsAg were measured in serum 8 weeks after primary immunization. Mice received 100 µg of HBsAg DNA or 3 µg of the alum-based protein HBsAg vaccine (GSK) IM.

Figure 13 shows that IM immunization of Balb / c mice with HBsAg DNA polarizes the behavior of peripheral T lymphocytes (spleen cells) to a cytokine profile with Th1 tendency. The left panel shows the results of the IFNγ ELISPOT test, and the right panel shows the results of the IL-10 ELISPOT test. The graph shows a comparison between the HBsAg CTL peptide and the HBsAg protein in the activation of T lymphocytes for cytokine production in both assays. Irrelevant peptides and proteins are also included as controls for specificity. Polyclonal responses for Concavalin A from T lymphocyte mitogens (With A) are shown for a maximum response for IL-10. The results for stimulation with A in the IFN-γ assay are not shown, since the points were too numerous to be accurately quantified. Both the peptide and the protein are capable of stimulating IFN-γ production in mice immunized with HBsAg DNA, but none produces significant IL-10 production, directly demonstrating a strong tendency of cytokines to Th1 in mice immunized with DNA. . Unimmunized mice did not produce detectable numbers of cytokine points on stimulation with the HBsAg protein peptide (data not shown).

Figure 14 illustrates the effect of heterologous immunization in Balb / c mice: Mice immunized with HBsAg DNA produce high levels of Ig in HBsAg specific serum after booster with a low dose of HBsAg IN liposomes. Mice were immunized with HBsAg DNA intramuscularly (100 µg at weeks 0 and 6) and boosted with a low dose of liposomes of HBsAg (3 µg / dose at week 12, n = 8 mice per group). The closed symbols show the responses of Ig antibodies in specific serum of total HBsAg before the IN booster. The closed symbols with cross hatching (+) show the Ig serum-specific Ig responses of total HBsAg 4 weeks after the IN booster.

Figure 15 illustrates the effect of heterologous immunization in Balb / c mice: mice immunized with HBsAg DNA produce high serum and vaginal IgA levels specific to HBsAg after booster with a low dose of HBsAg IN liposomes. Mice were immunized with HBsAg DNA intramuscularly (100 µg at weeks 0 and 6) and were boosted with a low dose of HBsAg liposomes (3 µg / dose at week 12). The closed symbols show the total HBsAg specific IgA antibody responses just before the IN booster. Closed symbols with cross hatching (+) show HBsAg specific IgA responses 4-5 weeks after IN reinforcement. As a positive control, serum IgA responses are shown for mice immunized homologously at weeks 0 and 6 with 15 µg of HBsAg liposomes and tested two weeks later (cross hatched circles, +). All groups contained 8 mice.

Figure 16 shows that mucosal IgG is also generated in mice immunized with the heterologous immunization protocol. Balb / c mice received two primary IM immunizations with HBsAg DNA (100 µg each time) followed by IN immunization with HBsAg liposomes (15 µg), empty liposomes, or left untreated (n = 5 per group). Four weeks after the final immunization, mice were sacrificed and pulmonary and vaginal washes were measured. HBsAg specific IgG titers were obtained in the sample combinations using an antigen specific sandwich ELISA assay. Only mice that received the combination of HBsAg DNA and HBsAg IN liposomes generated mucosal antigen-specific IgG responses above the background levels observed in untreated mice.

Figure 17 shows the proportions of IgG1: IgG2a in the serum of individual Balb / c mice after a heterologous immunization protocol consisting of IM priming with HBsAg DNA (100 µg on weeks 0 and 6) and liposome reinforcement of HBsAg (3 µg at week 16). Serum samples were taken four weeks after reinforcement. All mice showed an immune response with a tendency to Th1 (IgG1: IgG2a ratio of <0.5). These results indicate that the response with Th1 tendency that has been observed after the only immunization with HBsAg DNA is preserved even after administration of HBsAg liposomes, which promote Th2 tendency responses in Balb / c mice (compare with Figure 11).

Figure 18 illustrates avidity indices of HBsAg specific serum in Balb / c mice immunized by homologous immunization protocols (left panel) or heterologous immunization protocols (right panel). Mice were immunized at weeks 0 and 4 (homologous immunization) or primed at weeks 0 and 4, and boosted at week 8 (heterologous immunization). The avidity of the antibodies was measured in serum obtained two weeks after the last immunization. The dashed line shows the maximum avidity values that are possible in this quantitative test. The heterologous immunization protocol generates IgG antibodies in HBsAg specific serum with the highest avidity. Homologous immunization with Ag IN liposome of the same experiment is shown in the right panel for comparison.

Figure 19 illustrates representative results of a CTL destruction assay in vivo. Left panel: splenic cells of untreated Balb / c mice. Right panel: splenic cells of Balb / c mice that received the heterologous immunization regimen (2 x HBsAg IM DNA + 1 x HBsAg IN liposomes). All mice received a mixture of syngeneic splenic cells without pulsing with CFSEbajo and syngeneic spleen cells pulsed with HBsAg peptide with CFSEalto. 20 hours later, the spleens of the receptors were cultured and analyzed to obtain CFSE fluorescence (x-axis) by flow cytometry. Region 1 is selected in non-fluorescent spleen (receptor) cells. Region 2 is selected in cells (donor without pressing) with CFSEbajo. Region 3 is selected in cells with CFSEalto (donors pulsed with HBsAg peptides). 200,000 viable cells were analyzed. The disappearance of CFSEalto (R3) cells in the right panel indicates the specific destruction of HBsAg peptides of CTL transfusion target cells in immunized mice.

Figure 20 illustrates the clearance of HBsAg vaccinia virus (VV-HBsAg) from the lungs of mice that received the heterologous immunization regimen. Balb / c mice were immunized as indicated (treatment groups; n = 5 mice per group) at four week intervals. Mice received doses of 100 µg of HBsAg IM DNA and 15 µg of HBsAg IN liposomes. Mice were infected 14 days after booster with 2 x 105 PFU of VV-HBsAg administered in a volume of 50 µl intranasally. Mice were sacrificed on day 5 after exposure to the virus, viral titers were determined in lung homogenates using Vero cells. The values shown are the mean ± SD of four mice per group.

DETAILED DESCRIPTION OF THE INVENTION

I. Overview

The present invention is based at least partially on the surprising discovery of the Applicants that the combination of the heterologous immunization protocol with an antigen or immunogen mixed with liposomes successfully causes a large set of strong immune responses. Applicants have developed new procedures to elicit the desired immune responses to an antigen or immunogen. Two examples put

Highlight the strong immune responses that the methods of the invention are capable of eliciting in an animal. First, the methods of the invention can be used to elicit immune responses that protect animals from exposure to live pathogens, such as a live virus. Second, vaccination using the methods of the invention confer immunity to individuals who do not respond to currently available vaccines. In addition, the new procedures provided in this

The document achieves the desired immune responses without the need for any adjuvant other than liposomes, thus avoiding risks and complications associated with many adjuvants, especially bacterial toxins, such as cholera toxin (CT) and labile enterotoxin (LT ) in the heat of E. coli. Depending on the particular immunization protocol used, the procedures described herein are capable of inducing a wide range of immune responses that includes a combination of the following: strong antibody responses, T lymphocyte responses, such as CTL generation, and production of Th1 type cytokine and local immunity in mucosal sites.

Accordingly, the present invention provides a method for eliciting a desired immune response in an animal, comprising nucleic acids encoding an antigen and a secondary preparation comprising the antigen and liposomes to elicit the desired immune response in the animal. The procedure is particularly useful for eliciting an immune response in a host animal against a pathogen. In said context, the primary preparation comprises nucleic acid encoding a pathogen specific antigen and the secondary preparation comprises the pathogen specific antigen and liposomes. In certain embodiments, the pathogen is Hepatitis B Virus (HBV). The heterologous immunization regimens of the present invention include priming and boosting with different forms of HBsAg vaccines (eg, HBsAg IM DNA and HBsAg IN or IM liposomes) that result in stronger systemic immune responses and also the induction of local immunity in mucosal sites. In one embodiment, priming with an HBsAg DNA vaccine intramuscularly and strengthening with HBsAg liposomes intranasally induced potent responses of synergistic antibodies, activated CTLs and Th1-type cytokine profiles, and caused the production of IgA e Secretory IgG in mucosal sites. In another embodiment, priming with an HBsAg DNA vaccine intramuscularly and strengthening with HBsAg liposomes intranasally generated antigen-specific lung CTLs and clearance of the complete virus after intranasal exposure to a recombinant vaccinia virus expressing HBsAg (VV-HBsAg). The delivery of intranasal reinforcement was established as a determining factor for the generation of local immunity.

The present invention also provides a kit for eliciting an immune response in an animal. The kit comprises a first immunizing component comprising a nucleic acid sequence encoding an antigen, a second immunizing component comprising the antigen and liposomes, and an instruction for a user to administer to the animal the first immunizing component followed by administration of the second Immunizing component to cause an immune response in the animal.

The invention further provides a method for modulating immune responses in such a way that a desired immune response that leads to a Th1 response in a host animal can be elicited. The method comprises intramuscularly administering to the animal a primary preparation comprising a nucleic acid sequence encoding an antigen; and subsequently, administer to the animal a secondary preparation comprising the antigen and the liposome. "What shop to" It refers to the situation in which the observed immune response is closer to a Th1 or Th2 response compared to before immunization. In certain embodiments, the immunization will completely change from a Th2 response to a Th1 response. In other embodiments, immunization may not completely change from a Th2 response to a Th1 response, but instead results in a mixed response or a weaker Th2 response.

The present invention further provides a method for developing effective vaccines and vaccination protocols specifically directed to the most common portal of entry for microorganisms, the mucous surfaces of the body. The invention provides vaccines that can be more effective by encapsulating them in liposomes with a defined composition / size, and delivering them directly to the mucous surfaces. By combining encapsulated vaccines with liposomes with appropriate adjuvants and immunization regimens, immune responses can be adapted to provide more effective and specific vaccines. In certain embodiments, the ideal vaccine will generate a balanced or polarized immune response with T collaborators 1 (Th1) that also includes strong antibody responses, CTL generation and production of Th1 type cytokine, and local immunity at mucosal sites. In other embodiments, it may be possible to adapt the immune response to generate a polarized immune response of T collaborators 2 (Th2), which may be beneficial in preventing graft rejection in the host and in protecting against certain parasitic infections.

The present invention provides methods for determining the optimal conditions for encapsulation of various antigens (such as HBsAg) in liposomes (e.g., doses of HBsAg, liposome size, proportion of liposome: protein, and fresh liposomes versus lyophilized and reconstituted) to generate the strongest antibody responses. Serum antibody responses were compared in mice that received said liposome encapsulated antigens (HBsAg liposome) via intranasal (IN) and intramuscular (IM) administration. The invention also describes the effect of the serum antibody response after one administration (main) or two administrations (main and secondary) of the same form of antigen (homologous immunization). It was found that liposomes are both a very effective adjuvant and an administration vehicle for inducing immunity to HBsAg by intranasal or intramuscular routes. In one embodiment, antibody responses to the test HBsAg antigen reached or exceeded the levels observed in mice treated with the commercially available HBsAg vaccine (GlaxoSmithKline) that is currently used in humans. Selective distribution of antigen from the mucous surfaces of the nasal passages also stimulated local production of secretory IgA in the mucous secretions on all sampled mucous surfaces.

Therefore, the invention provides several vaccine administration protocols that promote different types of immune responses (antibody production, T-cell cytokine profiles, CTL cellular immunity and local immunity on mucosal surfaces). The rational combinations of these administration platforms The rational combinations of these distribution platforms allow the development of vaccines that are adapted to provide the best protection against specific pathogens in certain circumstances, for example, to favor immune responses of type Th1 or Th2, or a mixed / more balanced response.

In one aspect, the present invention provides methods and reagents for effectively eliciting immune responses in animals, especially mammals, against certain antigens, such as viral antigens. An outstanding feature of the invention relates to the use of protein antigens encapsulated in liposomes administered intranasally (IN) or intramuscularly (IM) to the host animal.

In certain embodiments, the present invention provides methods and reagents for generating more effective vaccines by encapsulating antigens in liposomes of a defined composition and size, and administering the resulting antigen-liposome preparation directly to the mucosal immune system. In other embodiments, the application of the specific administration platform technologies of the invention provides a rational combination of said liposome encapsulated vaccines with appropriate adjuvants and immunization regimens, to adapt the immune responses to provide more effective and specific vaccines ( improving the cell-mediated and humoral immune response, adapting the Th2 and mixed / balanced response of Th1 / Th2 to Th1, increasing IFN-γ secretion, induction of the high level of IgA, IgG and mucosal CTL).

II. Immunization Protocols

A component of the methods and kits of the present invention is the use of a "primary" immunization, which comprises the initial administration of one or more antigens to an animal, especially a human patient, in preparation for one or more subsequent administrations. of the same antigen. Specifically, the term "primary", as used herein, refers to a first immunization that uses an antigen that induces an immune response to the desired antigen and claims a higher level of immune response to the desired antigen after subsequent reinmunization with the same antigen when administered in the context thereof or a different vaccine delivery system.

Another component of the methods and kits of the present invention is the use of a "secondary immunization";

or a "booster", which refers to the administration of a composition that supplies the same antigen as encoded in the primary immunization. Sometimes, a booster refers to an anamnestic response, that is, an immune response in a previously sensitized animal. Multiple reinforcements can be administered, using the same or different amounts for each reinforcement. A booster that uses an antigen delivery system other than priming may be referred to as a "heterologous booster" while a booster that uses the same antigen delivery system as the bait can be referred to as a "homologous booster."

As an alternative to sequential administration described above, the primary preparation and the secondary preparation can be administered simultaneously, that is, substantially at the same time. The methods of the invention lead to potent synergistic effects between primary immunization and secondary immunization in terms of immune responses that the procedures are capable of causing in an animal. As a result, the methods of the invention allow to provoke a desired level of immune response, in which each of the primary preparation and the secondary preparation, when administered alone to the animal, is insufficient to achieve the desired level of immune response. In certain particular embodiments, heterologous booster immunization generates mucosal CTLs (CD8 + IFNγ + phenotype and destruction of CTL in vivo), and provides complete protection against exposure to live pathogens, such as live HBV exposures, as shown by Applicants in the illustrative examples.

The effects of the immune response on the host animal can be assessed by various assays, including humoral and cellular immune responses. The humoral immune response includes titers (Ig) of total antigen specific antibodies in serum or on mucosal surfaces; titers of HBsAg specific antibodies in serum or mucosal surfaces; titres of isotypes and / or subtypes of specific antibodies that include IgG, IgA, IgG1 and IgG2a; proportion of IgG1 and IgG2a. The cellular immune response includes the phenotype and activity of cytotoxic T lymphocytes (CTL). The cellular immune response also includes the secretion of cytokines characteristic of Th1 responses, including IFN-γ, and secretion of cytokines characteristic of the Th2 response, including IL-10 and IL-4. Cytokines are detected directly by ELISPOT cytokine assays (i.e., IFN-γ and IL-10) and are deduced from proportions of IgG1: IgG2a (for example, Th1 response to Th2).

Each of the primary preparation and the secondary preparation can be administered by any of the following routes: subcutaneously, intramuscularly, intradermally, and mucosally. The primary preparation and secondary preparation can be administered by the same route, or by different routes. In certain preferred embodiments, the primary preparation is administered intramuscularly and the secondary preparation is administered intramuscularly or intranasally, resulting in not only strong systemic responses, but also the induction of local immunity at mucosal sites. In a particular embodiment, priming with an HBsAg DNA vaccine intramuscularly and boosting with HBsAg liposomes intranasally induced potent responses of synergistic antibodies, activated CTL and Th1-type cytokine profiles, and conserved IgA production. Secretory in mucosal sites. The administration of intranasal reinforcement was established as an important determining factor for the generation of local immunity.

Different intervals between primary administration and secondary administration can be used. Secondary administration can be done 2-8 weeks, preferably 4-6 weeks apart from the previous administration (initial or a previous reinforcement). Typically, a booster administered 4-6 weeks after initial administration is sufficient. The invention contemplates that the primary preparation or the secondary preparation,

or both, can be administered to the animal one or more times.

The administration or secondary administrations and the administration or initial administrations may be used the same or different amounts of antigen-liposome preparations, and the reinforcement and initial administrations may be administered through the same or different routes. The administration or initial administrations and the administration or secondary administrations can be used different amounts of DNA antigen administered according to different schedules, and different amounts of antigenoliposome preparations administered.

III. Antigens

As used herein, the terms "antigen" or "immunogenic", used interchangeably, are intended to include all peptide or protein sequences that are capable of inducing an immune response in the animal in question. The terms "antigen" or "immunogen" they include peptide or protein analogs of known or wild-type antigens, the analogs of which may be more soluble or more stable than the wild-type antigen, and which may also contain mutations or modifications that make the antigen more immunologically active or optimized for expression in certain types of cells (i.e. use of humanized codons). An antigen can also be a peptide in which particular amino acid substitutions have been made to a naturally occurring antigen that alter the structure of the protein, a portion of the naturally occurring antigen that includes known protective epitopes (i.e. CTL epitopes ), or a synthetic origin chain of known epitopes that may or may not be limited to a pathogen (multivalent vaccine).

Also useful are additional peptides or proteins that have homologous sequences with an amino acid sequence of the desired antigens, in which the homologous antigen induces an immune response to the respective pathogens. Genes that are homologous to the sequence encoding the desired antigen should be interpreted to be included in the present invention, provided they encode a protein or polypeptide that has a biological activity substantially similar to that of the desired antigen.

The analogs of the antigens described herein may differ from naturally occurring proteins or peptides by the conservative amino acid, the differences in the sequence or by modifications that do not affect the sequence, or by both. For example, changes can be made in conservative amino acids, which although they modify the primary sequence of the protein or peptide, normally do not alter its function. Modifications (which normally do not alter the primary sequence) include in vivo or in vitro chemical derivation of polypeptides, for example, acetylation or carboxylation. Also included as antigens are glycosylation modified proteins, for example, those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in additional processing steps; for example, by exposure of the polypeptide to enzymes that affect glycosylation, for example, the glycosylation or deglycosylation enzymes of mammals. Also included as antigens according to this invention are sequences that have phosphorylated amino acid residues, for example, phosphotyrosine, phosphoserine or phosphotreonin. Polypeptides that have been modified using ordinary molecular biology techniques are also included as antigens in order to improve their resistance to proteolytic degradation or to optimize solubility properties. Analogues of such polypeptides include those containing residues other than naturally occurring L-amino acids, for example, Daminoacids or synthetic amino acids that are not naturally occurring. The antigens of the invention are not limited to the products of any of the specific exemplary processes listed herein.

An antigen can be a full length antigen, an immunogenic fragment thereof, or an epitope obtained from the antigen. In certain embodiments, the pathogen specific antigen in the secondary preparation may be in the form of an attenuated or destroyed pathogen. Effective antigens also include surface antigens of these pathogens.

The term "epitope", as used herein, refers to a sequence of at least about 3 to 5, preferably about 5 to 10 or 15, and no more than about 1,000 amino acids (or any integer between themselves), which define a sequence that by itself or as part of a larger sequence, binds to an antibody generated in response to said sequence or stimulates a cellular immune response. The term &quot; epitope &quot; it includes sequences identical to the native sequence, as well as modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature). The antigens used in the invention may comprise a single epitope, such as, for example, a single CTL epitope.

The antigens encoded by nucleic acids in the primary preparation and the antigens in the secondary preparation preferably have overlapping epitopes. In certain embodiments, the two antigens may be identical to each other. Alternatively, the two antigens may have overlap but different sets of epitopes. As an illustrative example, in the HBV vaccination protocol, DNA HBsAg can be used in the primary preparation, and the secondary preparation can be inactivated by the HBV. By way of another illustrative example, the primary preparation may be a minigene encoding a single epitope found in the HBsAg, and the secondary preparation may comprise the HBsAg antigen, or vice versa.

The following are illustrative examples of antigens that can be used in the present invention.

The antigens can be obtained from HIV-1, (such as tat, nef, gp120 or gp160, gp40, p24, gag, env, vif, vpr, vpu, rev), human herpes virus, such as gH, gL gM gB gC gK gE or gD or derivatives thereof or immediate early protein, such as ICP27, ICP47, IC P4, ICP36 of HSV1 or HSV2, cytomegalovirus, especially human, (such as, gB or derivatives thereof), Epstein virus Barr (such as, gp350 or derivatives thereof), Varicella Zoster Virus (such as, gpI, II, III and IE63), or from a hepatitis virus, such as hepatitis B virus (for example, antigen of Hepatitis B surface or Hepatitis core antigen or pol), hepatitis C virus antigen and hepatitis E virus antigen, or from other viral pathogens, such as paramyxovirus: Respiratory Syncytial Virus (such as, proteins F and G or derivatives thereof), or antigens of the parainfluenza virus, measles virus, mumps virus, human papilloma (for example, HPV6, 11, 16, 18, for example, L1, L2, E1, E2, E3, E4, E5, E6, E7), flavivirus (for example, Yellow Fever Virus, Dengue Virus , Tick Encephalitis Virus, Japanese Encephalitis Virus) or influenza virus cells, such as HA, NP, NA or M proteins, or combinations thereof, or antigens obtained from bacterial pathogens, such such as Neisseria spp, including N. gonorrhea and N. meningitidis for example, transferrin binding proteins, lactoferrin binding proteins, PilC, adhesins); S. pyogenes (for example, M proteins or fragments thereof, C5A protease), S. agalactiae, S. mutans; H. ducreyi; Moraxella spp, including M. catarrhalis, also called Branhamella catarrhalis (for example, adhesins and invasins of high and low molecular weight); Bordetella spp, including B. pertussis (for example, pertactin, pertussis toxin or derivatives thereof, filamentous hemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B. bronchiseptica; Mycobacterium spp., Including M tuberculosis (for example, ESAT6, Antigen 85A, -B or -C, MPT 44, MPT59, MPT45, HSP10, HSP65, HSP70, HSP 75, HSP90, PPD 19 kDa [Rv3763], PPD 38 kDa [Rv0934]), M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Legionella spp, including L. pneumophila; Escherichia spp, including enterotoxic E. coli (for example, colonization factors, heat labile toxins or derivatives thereof, heat stable toxins or derivatives thereof), enterohemorrhagic E. coli, enteropathogenic E. coli (for example, toxin similar to shiga toxin or derivatives thereof); Vibrio spp, including V. cholera (for example, cholera toxin or derivatives thereof); Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y. enterocolitica (for example, a Yop protein), Y. pestis, Y. pseudotuberculosis; Campylobacter spp, including C. jejuni (for example, toxins, adhesins and invasins) and C. coli; Salmonella spp, including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp., Including L. monocytogenes; Helicobacter spp, including H. pylori (for example, urease, catalase, vacuolizing toxin); Pseudomonas spp, including P. aeruginosa; Staphylococcus spp., Including S. aureus, S. epidermidis; Enterococcus spp., Including E. faecalis, E. faecium; Clostridium spp., Including C. tetani (for example, tetanus toxin and derivatives thereof), C. botulinum (for example, botulinum toxin and derivatives thereof), C. difficile (for example, Clostridium A toxins or B and derivatives thereof); Bacillus spp., Including B. anthracis (for example, botulinum toxin and derivatives thereof); Coryne-bacterium spp., Including C. diphtheriae (for example, diphtheria toxin and derivatives thereof); Borrelia spp., Including B. burgdorferi (for example, OspA, OspC, DbpA, DbpB), B. garinii (for example, OspA, OspC, DbpA, DbpB), B. afzelii (for example, OspA, OspC, DbpA, DbpB), B. andersonii (for example, OspA, OspC, DbpA, DbpB), B. hermsii; Ehrlichia spp., Including E. equi and the agent of the Human Granulocytic Erliquiosis; Rickettsia spp, including R. rickettsii; Chlamydia spp., Including C. trachomatis (for example, MOMP, heparin-binding proteins), C. pneumoniae (for example, MOMP, heparin-binding proteins), C. psittaci; Leptospira spp., Including L. interrogans; Treponema spp., Including T. pallidum (for example, rare outer membrane proteins), T. denticola, T. hyodysenteriae; or obtained from parasites, such as Plasmodium spp., including P. falciparum; Toxoplasma spp., Including T. gondii (for example, SAG2, SAG3, Tg34); Entamoeba spp., Including

E. histolytica; Babesia spp., Including B. microti; Trypanosoma spp., Including T. cruzi; Giardia spp., Including

G. lamblia; Leshmania spp., Including L. major; Pneumocystis spp., Including P. carinii; Trichomonas spp., Including T. vaginalis; Schisostoma spp., Including S. mansoni, or obtained from yeasts, such as Candida spp., Including C. albicans; Cryptococcus spp., Including C. neoformans.

Other preferred specific antigens for M. tuberculosis are, for example, Rv2557, Rv2558, RPFs: Rv0837c, Rv1884c, Rv2389c, Rv2450, Rv1009, aceA (Rv0467), PstS1, (Rv0932), SodA (Rv3846), Rv2031c 16Da 16v, Tb201c 16Da Ra12, Tb H9, Tb Ra35, Tb38-1, Erd 14, DPV, MTI, MSL, mTTC2 and hTCC1 (WO 99/51748). Proteins for M. tuberculosis also include fusion proteins and variants thereof in which at least two, preferably three M. tuberculosis polypeptides are fused into a larger protein. Preferred fusions include Ra12-TbH9-Ra35, Erd14-DPV-MTI, DPV-MTI-MSL, Erd14-DPV-MTI-MSI-mTCC2, Erd14-DPV-MTI-MSL, DPV-MTI-MSL-mTCC2, TbH9- DPV-MTI (WO 99/51748). The most preferred antigens for Chlamydia include, for example, High Molecular Weight Protein (HWMP) (WO 99/17741), ORF3 (EP 366412), and putative member proteins (Pmps). Other Chlamydia antigens of the vaccine formulation may be selected from the group described in WO 99/28475.

Preferred bacterial vaccines comprise antigens obtained from Streptococcus spp, including S. pneumoniae (PsaA, PspA, streptolysin, choline binding proteins) and antigen protein pneumolysin (Biochem Biophys Acta, 1989, 67,1007; Rubins and col., Microbial Pathogenesis, 25, 337-342), and detoxified derivatives thereof (WO 90/06951; WO 99/03884). Other preferred vaccines comprise antigens obtained from Haemophilus spp., Including H. influenzae type B (for example, PRP and conjugates thereof), non-typable H. influenzae, for example, OMP26, high molecular weight adhesins, P5, P6, protein D and lipoprotein D, and peptides obtained from fimbrine and fimbrine (US 5,843,464) or multiple copy variants or fusion proteins thereof.

Antigens that can be used in the present invention may additionally comprise antigens obtained from parasites that cause malaria. For example, the preferred antigens of Plasmodia falciparum include RTS, S and TRAP. RTS is a hybrid protein that comprises substantially all C-terminal portions of the P-falciparum circumsporozoite (CS) protein bound through four amino acids of the preS2 portion of the Hepatitis B surface antigen to the surface antigen (S) Hepatitis B virus. Its complete structure is described in International Patent Application No. PCT / EP92 / 02591, published under No. WO 93/10152 claiming priority over United Kingdom Patent Application No. 9124390.7. When expressed in yeast, RTS is produced as a lipoprotein particle, and when co-expressed with HBV S antigen produces a mixed particle known as RTS, S. TRAP antigens are described in International Patent Application No. PCT / GB89 / 00895, published under No. WO 90/01496. A preferred embodiment of the present invention is a Malaria vaccine in which the antigen preparation comprises a combination of the RTS, S and TRAP antigens. Other plasmodia antigens that are possible candidates to be components of a multi-stage malaria vaccine are P. faciparum MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, Sequestrina, PfEMPI, Pf332, LSA1, LSA3, STARP, SAUCE, PfEXP1, Pfs25, Pfs28, PFS27 / 25, Pfs16, Pfs48 / 45, Pfs230 and their analogs in Plasmodium spp.

The vaccines of the present invention can also be used for the prophylaxis or therapy of chronic disorders in addition to allergic or infectious diseases. Such chronic disorders are diseases such as asthma, atherosclerosis and Alzheimer's, and other autoimmune diseases. Vaccines can also be considered for use as a contraceptive.

The relevant antigens for the prophylaxis and therapy of patients susceptible to or suffering from Alzheimer's neurodegenerative disease are, in particular, the 39-43 N-terminal amino acid fragment (AB, the amyloid precursor protein and smaller fragments. This antigen It is described in International Patent Application No. WO 99/27944 - (Athena Neurosciences).

Potential autoantigens that can be included as vaccines for autoimmune disorders or as a contraceptive vaccine include: cytokines, hormones, growth factor or extracellular proteins, more preferably a 4-helic cytokine, more preferably IL13. Cytokines include, for example, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL20, IL21, TNF, TGF , GMCSF, MCSF and OSM. The 4-helices cytokines include IL2, IL3, IL4, IL5, IL13, GMCSF and MCSF. The hormones include, for example, luteinizing hormone (LH), follicle stimulating hormone (FSH), chorionic gonadotropin (CG), VGF, GHrelin, agouti, agouti-related protein and neuropeptide Y. Growth factors include, for example, VEGF

IV. Primary preparation and secondary preparation

The nucleic acids used in the primary preparation may be RNA or DNA that include DNA, synthetic DNA or cDNA. Preferably, the nucleotide sequence is a DNA sequence and more preferably, a cDNA sequence. In order to obtain the expression of the antigenic peptide in mammalian cells, it is necessary for the nucleotide sequence to encode the antigenic peptide that will be present in an appropriate vector system. By "appropriate vector", as used herein, refers to any vector that allows the antigenic peptide to be expressed in a mammal in amounts sufficient to elicit an immune response.

For example, the selected vector may be a plasmid, a phagemid or a viral vector. The vector may comprise a promoter and polyadenylation / transcription termination sequence arranged in the correct order to obtain the expression of antigenic peptides. The construction of vectors that include these components and other optional components, such as enhancers, restriction enzyme sites and selection genes, such as antibiotic resistance genes, is well known to those skilled in the art and is explained in detail in Maniatis et al "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor Press, Volumes 1-3, 2nd Edition, 1989.

A vector carrying nucleic acids encoding an antigenic peptide can be administered in a variety of ways. This is possible for the vector that is administered in a simple form (which is like the nude nucleotide sequence that is not in association with liposome formulations, with viral vectors or proteins that facilitate transfection) suspended in an appropriate medium, for example , a buffered saline solution, such as PBS, and then injected intramuscularly, subcutaneously, intradermally or mucosally. This is additionally possible for vectors to be encapsulated by liposome particles or in co-glycolide polylactide (PLG) (25) for nasal or pulmonary administration.

This is also possible, according to an embodiment of the invention, for intradermal administration of the vector, preferably by the use of gene gun administration techniques (particularly particle bombardment). Such techniques may involve coating the vector on gold beads that are then administered at high pressure in the epidermis, such as, for example, as described in Haynes et al. J. Biotechnology 44: 37-42 (1996).

Recombinant viral vectors can also be used to deliver DNA antigens. The advantages of this approach include the abundant expression of DNA-encoded proteins in multiple cell types, strong improvement of CTL responses and the ability of the virus to encode multiple genes. The vaccinia virus (which includes the modified Ankara virus) and adenovirus (non-replicating) are two popular viruses used for vaccine development (Im and Hanke, Expert, Rev. Vaccines 3: S89-S97 (2004); Basak et al. , Viral Immunol. 17: 182-196 (2004)).

Recent modifications of DNA vaccines include the development of minigenes encoding individual CTL epitopes, the use of signals directed to gene coding to allow a more efficient presentation of epitopes by the MHC pathway and the generation of secreted proteins to the path. MHC target class II (Doria-Rose and Haigwood, Methods 31: 207-216 (2003); Leifert et al., Immunol. Rev. 199: 40-53 (2004)).

The primary and secondary preparations are administered in an amount such that they were prophylactic and therapeutically effective. The exact amount can vary considerably depending on the species and the weight of the animal to be immunized, the route of administration, the potency and the dose of the primary and secondary preparations, the nature of the pathology to be treated or protected. versus, the ability of the subject's immune system to produce an immune response and the degree of protection or therapeutic effect desired. Based on these variables, a veterinarian can easily determine the appropriate dosage level.

It is contemplated that the methods and kits of the invention can also be practiced with the addition of one or more adjuvants known in the art. An adjuvant is a substance or procedure that increases specific immune responses to antigens by modulating the activity of immune cells. Exemplary adjuvants include salt-based adjuvants, such as alum salts, bacterial-derived adjuvants such as lipopolysaccharides and bacterial toxins, adjuvant emulsions that allow slow antigen release, agonist antibodies to co-stimulate molecules, Freunds adjuvant, dipeptides muramil, and recombinant / synthetic adjuvants. Alum is the most common salt-based adjuvant used to stimulate immune responses to protein vaccines and is the only adjuvant approved for human use in the United States (Alving, Vaccine 20 (3): 556-S64 (2002); Hunter, Vaccine 20 (3): S7-12 (2002)). However, alum favors Th2-prone responses and does not stimulate cell-mediated immunity. Mucosal immunity can be induced through the use of bacterial toxins, such as cholera toxin (CT) and heat labile enterotoxin E. coli (LT), however, the safety of these adjuvants is questionable (Alving, Vaccine 20 ( 3): S56-S64 (2002); Hunter, Vaccine 20 (3): S7-12 (2002)). The development of newer and safer adjuvants has recently focused on the stimulation of particular immune response trajectories. Co-administration of cytokines, such as interferon-γ and granulocyte-macrophage colony stimulating factor (GM-CSF), has shown promise in stimulating cellular immune responses (reviewed in (Petrovsky and Aguilar, Immunol. Cell Biol. 82: 488496 (2004) .However, high levels of cytokines can cause cytotoxicity, and dosage regimens have to be carefully modulated.The administration of cytokines is particularly promising for DNA vaccination in which the genes that encoding both the cytokine and the antigen can be expressed simultaneously by the same vector. Additional adjuvants to be explored include those that target the toll signaling pathway. The CpG DNA motifs normally found in bacterial DNA are potent. activators of cellular immune responses, and new generation DNA-based vaccines often encode multiple CpG motifs (reviewed and n (Petrovsky and Aguilar, Immunol. Cell Biol. 82: 488-496 (2004)).

V. Pathogens

The term "pathogen", as used herein, refers to any virus, bacteria and parasites that are capable of causing an infection in a host animal.

The present invention can be used to effectively immunize host animals against a range of pathogens. In light of the teachings herein, one skilled in the art will appreciate that the methods and compositions of the invention are applied to a wide range of viruses, such as, for example, reovirus, RNA virus, DNA virus and virus. encapsulated In one embodiment, the present invention is applied to reovirus. In another embodiment, the present invention is further applied to retroviruses (retroviridae). In yet another embodiment, the present invention is applied to lentivirus, including a primate lentivirus group. In a further embodiment, the present invention is applied to Human Immunodeficiency Virus (HIV), Human Immunodeficiency Virus type 1 (HIV-1), Hepatitis B virus (HBV), Hepatitis A Virus (HAV) ), Hepatitis C Virus (HCV) and Human T Lymphocyte Leukemia Virus (HTLV).

Although not intended to be limiting, retroviruses are relevant include: type C retrovirus that causes pike lymphosarcoma, type C retrovirus, that infects minks, goat lentiviruses that infect sheep, Infectious Anemia virus Equine (EIAV), type C retrovirus, which infects pigs, Avian Sarcoma Leukosis virus (ALSV), feline leukemia virus (FeLV), feline AIDS virus, leukemia virus bovine (BLV), simian leukemia virus (SLV), simian immunodeficiency virus (SIV), human T lymphocyte leukemia virus type I (HTLV-I), virus type II human T lymphocyte leukemia (HTLV-II), human immunodeficiency virus type 2 (HIV-2) and human immunodeficiency virus type 1 (HIV-1).

The method and compositions of the present invention are further applied to RNA viruses, including viruses from negative strains of RNAs and viruses from positive strains of RNAs. These virus strains positive for siRNA include the Hepatitis C virus (HCV). In a preferred embodiment, the present invention is applied to mononegavirals, including filovirus. Filoviruses additionally include Ebola viruses and Marburg viruses. Other RNA viruses include picomaviruses, such as enteroviruses, polioviruses, coxsackieviruses and hepatitis A viruses, caliciviruses, which include Norwalk-like viruses, rabdoviruses, including rabies virus, togaviruses, including alphaviruses, Semliki viruses. Forest, denguevirus, yellow fever virus and rubella virus, orthomyxovirus, including influenza A, B and C influenza virus, rotavirus, bunyavirus, including Rift Valley fever virus and hantavirus , filoviruses, such as Ebola virus and Marburg virus, and paramyxoviruses, including mumps virus and measles virus. Other viruses that can be treated with the methods of the present invention include Herpes Simplex virus type I (HSV1), Herpes Simplex virus type II (HSV2), Bacillus anthracis, respiratory syncytial virus, Coronavirus (pathogen for SARS) and virus of paw and mouth disease (FMDV). Additional pathogens that can be treated include Chlamydia and malaria-causing parasites (including Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale).

The methods and kits according to the present invention can be used in relation to the prophylactic or treatment procedures of all animals, including, for example, domestic animals, laboratory animals, farm animals, wild animals in captivity and, more preferable, human beings.

The methods and kits of the invention are useful for therapeutic vaccination. The goal of therapeutic vaccination is to direct immune responses in an infected individual to eradicate the cells that are infected with the virus. Viruses that establish chronic infections without stimulating strong immune responses require the generation of new therapeutic vaccination strategies (reviewed in (Berzofsky et al., J. Clin. Invest. 114: 450-462 (2004)). Papillomavirus human (HBV), hepatitis B and C viruses (HBV, HCV), herpes virus and human immunodeficiency virus (HIV) are several of the viruses that establish long-term infections.The development of therapeutic vaccines for The virus has focused on the activation of CTL to recognize and destroy infected cells Applicants have shown that the methods of the invention are effective in improving cellular immune responses, making them suitable for providing therapeutic vaccination. procedures can be further improved by the inclusion of cytokine adjuvants and CpG motifs that have been particularly promising in the development of anti-cancer vaccines. erosas (Belardelli et al., Cancer Res. 64: 6827-6830 (2004)).

The vaccines of the present invention are particularly suitable for the immunotherapeutic treatment of diseases, such as chronic conditions, but also for the therapy of persistent infections. Accordingly, the vaccines of the present invention are particularly suitable for immunotherapy of infectious diseases, such as Tuberculosis (TB), HIV infections, such as AIDS and HBV infections.

SAW. Liposome Preparation

The present invention also provides a liposome-based antigen administration platform technology for vaccine applications through mucosal sites. The invention combines liposome encapsulation technology, specific administration to mucosal sites, and the use of adjuvants and heterologous immunization protocols to achieve a balanced or mixed Th response.

Liposomes have several potential advantages as administration platforms for vaccines. Encapsulation of antigens in liposomes sequesters these antigens, therefore, liposomes serve as an antigen reservoir capable of releasing antigen in a sustained manner. In addition, liposomes are biocompatible and biodegradable, and low in toxicity and immunogenicity. When they have the appropriate size (for example,> 0.2 µm to 5 µm), liposomes are selectively collected by cells that have antigen in the body, and have the potential to induce both humoral and CTL antibody responses. Liposomes serve as an antigen carrier / vehicle, as well as being an adjuvant that can be administered repeatedly without toxicity or immunogenicity.

Liposomes are structures composed of a bilayer membrane composed of phospholipids of biological origin

or synthetic, usually spherical. Liposomes form naturally when phospholipids or lipids come into contact with water. The structure of the liposomes can be manipulated by procedures for their formation in the laboratory, including the supply of energy in the form of heat, sound energy, freezing and thawing cycles, or shear forces. Because liposomes have the characteristics of biological membranes, they can be engineered in the laboratory to contain a variety of molecules of relevant biological and therapeutic complexes, including proteins. The bilayer phospholipid membrane of the liposomes separates and protects the material trapped in the inner aqueous core from the outside. Both water soluble and insoluble substances can be trapped in the different compartments, the aqueous core and two-layer membrane, respectively, of the same liposome. The physical and chemical interaction of these substances can be eliminated since the substances are in these different compartments.

The liposomes used with the methods and kits of the present invention can be prepared using any method known in the art. These liposomes may have an average diameter of about 0.5-5 micrometers, although liposomes of 0.2-8 micrometers may also be useful. In certain embodiments, the liposomes that may be useful in the methods and kits of the invention are "long circulating liposomes". (also known as "sterically stabilized liposomes"), which are liposomes comprising one or more specialized lipids that, when incorporated into the liposomes, result in an improved circulation life in relation to liposomes lacking such specialized lipids. Examples of long circulating liposomes known in the art include those in which the liposome (A) comprises one or more glycolipids, such as GM1 monosialoganglioside or (B) comprises one or more lipids derived with one or more hydrophilic polymers, such as a rest of polyethylene glycol (PEG). Without wishing to be bound by theory, at least for long circulating liposomes containing gangliosides, sphingomyelin or lipids modified with PEG, the improved circulation half-life of these liposomes is obtained from a reduced reuptake in reticuloendothelial system (RES) cells (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

A. Liposome comprising glycolipids: Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) indicated the ability of GM1 monosialoganglioside, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were presented by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). Pat. No. 4,837,028 and published PCT Application WO 88/04924, both of Allen et al., describe liposomes comprising (1) sphingomyelin and (2) the GM1 ganglioside or a galactocerebroside sulfate ester. Pat. US 5,543,152 (from Webb et al.) describes liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimiristoylphosphatidicoline are described in published PCT application WO 97/13499 (from Lim et al.).

B. Liposomes Modified with Hydrophilic Polymers: Many liposomes comprising lipids modified with one or more hydrophilic polymers, and methods for preparing them, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a non-ionic detergent, 2C1215G, which contained a PEG moiety. Illum et al. (FEBS Letters, 1984, 167, 79) indicated that the hydrophilic coating of polystyrene particles with polymeric glycols results in significantly improved half-lives in blood. Synthetic phospholipids modified by the binding of carboxylic groups of polyalkylene glycols (for example, PEG) and liposomes comprising said phospholipids are described by Sears (US Pat. No. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Letts., 1990, 268, 235) described experiments demonstrating that liposomes comprising PEG-modified phosphatidylethanolamine (PE) or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended these observations to other phospholipids modified with PEG, for example, DSPE-PEG, formed from the chemical binding of PEG to DSPE (distearoylphosphatidylethanolamine).

Liposomes having covalent binding to PEG moieties on their outer surface are described in European Patent No. 0 445 131 B1 and published PCT application WO 90/04384 by Fisher. Liposome compositions containing 1-20 mol% (mol%) of PE modified with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. No. 5,013,556 and 5,356,633) and Martin et al. (United States Pat. No.

5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising several different lipid-polymer conjugates are described in published PCT application WO 91/05545 and Pat. No. 5,225,212 (both by Martin et al.) and in published PCT application WO 94/20073 (Zalipsky et al.). Liposomes comprising ceramide lipids modified with PEG are described in published PCT application WO 96/10391 (Choi et al.). Pat. No. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe liposomes containing PEG that can be modified on their surfaces with functional moieties.

C. Liposomes Containing DMPG: Various liposomes comprising dimiristoylphosphatidylglycerol (DMPG) have been described. Generally, however, such liposomes comprise DMPG is a mole% of about 10% or greater (see, for example, Akhtar et al. (Nucl. Acids Res., 1991, 19, 5551; Yachi et al. (Biopharm Drug Dispos., 1996, 17, 699; and Farmer et al. (Meth. Enz., 1987, 149, 184). Liposomes having 3% DMPG in mol have been described, but said liposomes included a component (in in particular, a phosphatidylcholine derivative) which is not found in the liposomal compositions of the present invention Such components of phosphatidylcholine derivatives include, for example, 10% distearoylphosphatidylcholine in mol (DSPC) (Brodt et al., Cancer Immunol. Immunother ., 1989, 28, 54) or 7% dimiristoylphosphatidylcholine in mol (DMPC) (Perez-Soler et al., J. Nuclear Med., 1985, 26, 743; Wasan et al., Antimicrobial Agents and Chemotherapy, 1993, 37, 246; and Li et al., Oncology Res., 1995, 7, 611).

The liposome preparation may be freshly prepared or lyophilized for long-term storage. Both preparations can be used with comparable efficacy. The liposomes used in the methods of the invention may all be the same (eg, same compositions or same size), or include more than one type of liposome.

In certain embodiments, commercially available liposomes can be used. For example, liposomes can be prepared by contracting from Northern Lipids Inc. (Vancouver, BC), a Contracted Research Organization that specializes in the development of lipid-based liposome formations. In some other embodiments, liposomes of various sizes can be prepared using the methodology described below. The resulting liposomes, depending on the specific preparation protocols, typically have a size of 4 µm, 1 µm or 0.2 µm by passing the preparations through a microfluidizer. In summary, liposomes were prepared at the following lipid concentrations: 7/3 / 0.5% in mol of phosphatidylcholine / cholesterol / dicetyl phosphate. An antigen, such as HBsAg, is incorporated into multilamellar liposomes at various concentrations for the immune response assay. An average of 60% of HBsAg was incorporated into the liposomes in the examples indicated below. Liposome size was also measured with a Submicron N4 MC particle size analyzer (Coulter Electronics). A representative profile of liposomes with a size of 4 µm is shown in Figure 1. In one experiment, the liposome: protein ratio decreased to 1/2 and usually 1/3 of the content was used to encapsulate 15 µg of HBsAg. In certain embodiments, antigen preparations can be lyophilized for storage and then reconstituted before use. Both size and potential ζ (zeta, a load measure) can be measured before use. The potential ζ was determined using a potential analyzer ζ Zeta-Puls (Brookhaven Instruments). The lipid: antigen ratio may vary in some preparations in order to determine the importance of this ratio in immune responses to the specific antigen (eg, HBsAg).

A. Basic procedure for liposomes with a size range of 2-4 µm.

The liposomes of the subject invention were prepared at the following lipid concentrations: 7/3 / 0.5% by mole of phosphatidylcholine / cholesterol / dicetyl phosphate. Dicethyl phosphate was dissolved in chloroform plus 5% ethanol, sonicated and then phosphatidylcholine and cholesterol were added. The lipids were dried on a Labconco rotary evaporator for one hour and traces of chloroform were removed by lyophilization with a Freezone 4.5 Freeze-dried System overnight. The lipid film was hydrated with antigen, such as the Hepatitis B surface antigen (HBsAg), at a concentration of 125-300 µg / ml in 10 mM HEPES buffer, 150 mM NaCl, pH 7.4 (HBS) and filtered with 0.2 µm of nylon filter. The mixture was vortexed thoroughly and allowed to stand for 1 hour, and then vortexed again to ensure the formation of multilamellar vesicles. Then, the liposomes were subjected to three cycles of freezing and thawing (1 cycle = freezing for one hour and defrosting for one hour at room temperature). Liposome size was measured with a Submicron N4 MD Particle Size Analyzer (Coulter Electronics). The ζ potential was measured using a ζ Zeta-Puls (Brookhaven Instruments) potential analyzer in 5 mM HEPES buffer HEPES, 1.0 mM NaCl, pH 7.4.

B. Liposomes with a size of 1.0 µm

The liposomes of the subject invention were prepared at the following lipid concentrations: 7/3 / 0.5% by mole of phosphatidylcholine / cholesterol / dicetyl phosphate. Dicethyl phosphate was dissolved in chloroform plus 5% ethanol, sonicated and then phosphatidylcholine and cholesterol were added. The lipids were dried on a Labconco rotary evaporator for one hour and traces of chloroform were removed by lyophilization with a Freezone 4.5 Freeze-dried System overnight. The lipid film was hydrated with antigen (eg, HBsAg) at a concentration of 125-300 µg / ml in 10 mM HEPES buffer, 150 mM NaCl, pH 7.4 and filtered with 0.2 µm of nylon filter. The mixture was vortexed thoroughly and allowed to stand for 1 hour, and then vortexed again to ensure the formation of multilamellar vesicles. Then, the liposomes were subjected to three cycles of freezing and thawing (1 cycle = freezing for one hour and defrosting for one hour at room temperature). After the third cycle, the liposomes were heated in a 50 ° C water bath and extruded through a polycarbonate filter with a pore size of 1.0 µm using an Avanti portable micro extruder. The size of the liposomes was measured as before.

C. Liposomes with a size of 0.2 µm

The liposomes of the subject invention were prepared at the following lipid concentrations: 7/3 / 0.5% by mole of phosphatidylcholine / cholesterol / dicetyl phosphate. Dicethyl phosphate was dissolved in chloroform plus 5% ethanol, sonicated and then phosphatidylcholine and cholesterol were added. The lipids were dried on a Labconco rotary evaporator for one hour and traces of chloroform were removed by lyophilization with a Freezone 4.5 Freeze-dried System overnight. The lipid film was hydrated with antigen (eg, HBsAg) at a concentration of 125-300 µg / ml in 10 mM HEPES buffer, 150 mM NaCl, pH 7.4 and filtered with 0.2 µm of nylon filter. The mixture was vortexed thoroughly and allowed to stand for 1 hour, and then vortexed again to ensure the formation of multilamellar vesicles. Then, the liposomes were subjected to three cycles of freezing and thawing (1 cycle = freezing for one hour and defrosting for one hour at room temperature). After the third cycle, the liposomes were heated in a 50 ° C water bath and extruded through a polycarbonate filter with a pore size of 1.0 µm, then with a pore size of 0.4 µm and finally 0.2 µm using an Avanti portable microextruder. Then, the size of the liposomes was measured as previously indicated.

D. Preparation of lyophilized liposomes.

The liposomes of the subject invention were prepared at the following lipid concentrations: 7/3 / 0.5% by mole of phosphatidylcholine / cholesterol / dicetyl phosphate. Dicethyl phosphate was dissolved in chloroform plus 5% ethanol, sonicated and then phosphatidylcholine and cholesterol were added. The lipids were dried on a Labconco rotary evaporator for one hour and traces of chloroform were removed by lyophilization with a Freezone 4.5 Freeze-dried System overnight. The lipid film was hydrated with antigen (eg HBsAg) at a concentration of 125-300 µg / ml in HBS with 100 mg / ml maltose, and filtered with 0.2 µm of nylon filter. The mixture was vortexed thoroughly and allowed to stand for 1 hour, and then vortexed again to ensure the formation of multilamellar vesicles. Then, the liposomes were subjected to three cycles of freezing and thawing (1 cycle = freezing for one hour and defrosting for one hour at room temperature). The size of the liposomes was measured and the liposomes were lyophilized with a Freezone 4.5 Freeze Drying System overnight. Lyophilized liposomes were reconstituted by vortexing with a corresponding amount of water to reach the necessary concentration of antigens.

VII. essays

The following tests may be necessary to perform certain steps of the invention.

In general, as one expert knows, many different but functionally equivalent tests can be used to achieve the same purpose. Therefore, none of the tests specifically described is limiting unless explicitly stated.

A. Enzyme-linked Immunoabsorbent Assays (ELISA)

Responses of anti-HBsAg specific antibodies, such as Ig, IgG, IgA, IgG1 and IgG2a in samples collected from immunized mice were assayed by ELISA. A three-layer ELISA (antibody that detects antigen-assay samples or conjugated HRP of Ig standards) was designed to test Ig, IgG, IgG1 and IgG2a antibodies. 50 µl of 10 µg / ml of pure HBsAg (Advanced Immune Chemical) were coated in 96-well ELISA plates. Serial dilutions of serum or secretory samples were added after the blocking procedure. Mouse anti-HR (H + L) goat-HRP, goat anti-mouse IgG-HRP, goat anti-mouse IgG1-HRP and goat anti-mouse IgG2a-HRP (SouthernBiotech) as detection antibodies for the Ig assay , IgG, IgG1e IgG2a specific for anti-HBsAg, respectively in samples. Serially diluted IgG, IgG1 and IgG2a standards were used to generate conventional ELISA curves for IgG, IgG1 and IgG2a antibodies, respectively. Five-layer capture ELISAs were used to test mucosal samples for mouse IgA and mouse IgG. 50 µl of a 4 µg / ml rat anti mouse IgA capture antibody was coated on ELISA plates to capture IgA antibody from serum or mucosal sites (fecal, vaginal, lung wash and saliva). 100 µl of 2 µg / ml of purified HBsAg was added for binding to the anti-HBsAg specific IgA antibody. A goat anti-HBsAg was used to specifically bind the HBsAg captured in the previous stage. Donkey anti goat IgA-HRP was the final detection antibody before the color development of the substrate-TMB. For the detection of mucosal IgG a rat anti mouse IgG antibody was used as a capture antibody, followed by HBsAg, goat anti-HBsAg, and goat anti goat IgG-HRP donkey at the same concentrations that were used in the ELISA of IgA

B. Measurement of the avidity of IgG antibodies

The avidity of HBsAg-specific IgG antibodies is evaluated using quantitative ELISA avidity assays. The assays were performed using conditions optimized for IgG antibodies, with the addition of the addition of the chaotropic urea reagent to interrupt the antigen-antibody interactions. To ensure consistent and accurate results, the relative amount of IgG in the sample dilutions analyzed is between 100 150 ng / ml, which is within the linear and quantitative portion of the standard curve. Diluted samples are first incubated in ELISA plates coated with HBsAg for 1 hour at room temperature to allow antigen-antibody binding to occur. The wells were washed and subsequently incubated with conventional ELISA buffer (PBS with 0.05% Tween-20 and 1% bovine serum albumin), or with varying concentrations of

urea in ELISA buffer for 15 minutes at room temperature. After further washing, the ELISA was performed as usual. Avidity indices (AI) are calculated according to the following formula: ng / ml of IgG in the absence of urea / ng / ml of IgG in the presence of urea.

C. Detection of IFN-γ and IL-10 secretory cells by ELISPOT assays (Enzyme Linked Immunoabsorption Assay)

Spleen or peripheral blood cells from immunized and untreated mice were cultured for the measurement of IFN-γ and IL-10 secretory cells by ELISPOT assays. ELISPOT assays were established using commercially available pairs of antibodies and reagents (BD Pharmingen) and Multiscreen-IP multi-well ELISPOT plates (Millipore, Hopkington, MA). In vitro cultures were stimulated with HBsAg peptide at a concentration of 10 µg / 2.5 x 106 cells per ml for 20-22 hours. Peptide-free cultures were used as negative controls, and cultures stimulated with anti-CD3 or Con A were used as positive controls. The ELISPOT plates of IFN-γ and IL-10 of this study were quantitatively analyzed by Cellular Technology Ltd. (Cleveland, OH) to analyze both point frequencies and point size using a digital plate reader and software designed to this end.

D. Cytotoxicity assays of CTL in vivo

In vivo CTL assays have recently been described in the literature (references: Estcourt, MJ et al. 2002. Int. Immunol. 14 (1): 31-37; Barber, DL et al. 2003. J. Immunol. 171: 27-31; Kumaraguru, U. et al. 2004. J. Immunol.

172: 3719-3724) to measure CTL activity in immunized animals. The in vivo CTL assay uses mice that have received two rounds of an HBsAg IM DNA vaccine followed by the preparation of liposomes object IN (for example, HBsAg liposome). After the sample collection for the measurement of antibody responses is completed (six weeks or more after the IN booster), mice receive a DNA booster to mobilize memory T lymphocytes to activated effector cells. 7-12 days later, the activity of the effector cells is quantified by determining their ability to specifically destroy pulsed targets with HBsAg peptides that are adopted adoptively intravenously to the mouse. Mice receive two populations of untreated mouse cells that are differently labeled with intravital CFSE dye (fluorescent emission in the FITC channel): CFSE cells that are not pulsed with peptide, and CFSEalto cells that are pulsed with immunodominant peptide L (d) -restricted HBsAg (28-39 HBsAg). The disappearance of the CFSEalto population pulsed with peptide with respect to the CFSE population without pressing is the measure of the specific destruction of peptides according to the formula shown below. The percentages of these two populations in the untreated receptor cohorts serve as an internal control for cell grafting. A summary of this essay is shown below.

Preparation of target cells:

Untreated splenocytes: Mark with CFSE low. Pulsed without peptide. Check marking by flow cytometry. Untreated splenocytes: Mark with CFSEalto. Pulsed with peptide. Check marking by flow cytometry. Mix of equal numbers of CFSEbajo and CFSEalto cells (1-1.5 x 107 cells each).

CTL assay in vivo:

Transfer cells to cohorts of both immunized and untreated mice intravenously. 20-24 hours later, harvest spleens and process by flow cytometry and ELISPOT assays. Proportion = Percentage of CFSE cells Low: CFSE cells high

Specific Lysis% = 1

Measurement of protective mucosal immunity after intranasal exposure to recombinant live vaccinia virus expressing HBsAg (VV-HBsAg)

The hepatitis B virus is highly species specific and only infects humans and some nonhuman primates. To avoid this problem, the recombinant virus expressing HBsAg was used for exposure to the live virus in mice to test the protective efficacy of HBV vaccination protocols. A genetically engineered vaccinia virus vector expressing the HBsAg antigen (VV-HBsAg) was used for exposure to mice, kindly provided by Dr. Bernard Moss, Laboratory of Viral Diseases, NIAID, NIH (Smith, Mackett and Moss. 1983. Nature 302: 490-495). The feasibility and usefulness of using the recombinant vaccinia virus that expresses pathogen antigens for exposure to the virus has recently been demonstrated in mice in several disease virus models, including hepatitis C and HIV (Pancholi, P. et al. 2004 J.

Virol 77 (1): 382-90; Marata, K. et al. 2003. Proc. Natl Acad. Sci. USA 100 (11); Belyakov, IM et al. 1998. Proc. Natl Acad. Sci USA 95 (4): 1709-14).

Untreated or immunized mice were infected intranasally with 2 x 105 PFU virus in a volume of 50 µl. Mice were sacrificed after 5 days, and viral titers in lung homogenates were determined. Pulmonary washes and splenic cells were also evaluated to assess the presence of CTL, defined by a CD8 + IFNγ + phenotype.

F. Detection of CTL using the CD8 + IFNγ + phenotype after intranasal exposure with VV-HBsAg live

CTLs can be defined by an intracellular IFNγ + CD8 + surface phenotype according to well-recognized and standardized protocols (for example, see reagents and kits offered by BD Biosciences, San Diego, CA). Using these reagents, the frequency of CTLs defined by the phenotype was determined by flow cytometry analysis. To do this, mononuclear cells recovered from the lung and spleen were activated with the CTL HBsAg peptide (28-39 HBsAg) for five hours in vitro, in the presence of a golgi inhibitor to prevent secretion of accumulated intracellular cytokines. Then, the surface of the cells was stained for CD8, permeabilized and stained for intracellular IFNγ according to the manufacturer's guidelines.

VIII. Examples

This invention is further illustrated by the following examples that should not be construed as limiting.

In the following examples, the hepatitis B virus surface antigen (HBsAg) was selected as an illustrative antigen for the liposome-based vaccines in question. Hepatitis B infection is a significant health risk and is one of the leading causes of death worldwide. It is known that the response of antibodies to HBsAg confers protection against infection by the live virus in 5-10% of human beings. HBsAg is a well characterized antigen that contains numerous epitopes of T and B lymphocytes that make it a suitable model antigen to demonstrate immune responses in mice. In addition, the recombinant hepatitis B virus protein and gene expression constructs are also available in the market, eliminating the requirement to develop expensive reagents. However, it should not be construed that the invention is limited to the HBsAg vaccine. On the contrary, the present invention has many applications in a wide range of pathogens, including various viruses and bacteria.

EXAMPLE 1: Effect of the Primary and Secondary Immunization Protocol on Antibody Responses

To immunize a host animal, such as CD1 or Balb / c mice, the primary immunization using the HBsAg liposome preparation in question was administered to the animal at week 0. At week 6, secondary booster immunization was administered. HBsAg liposome.

To optimize the vaccine administration platform, several experiments were performed using different immunization strategies, as described in Examples 1-5. In this series of experiments, the liposomes were 4 µm in size, a size that was indicated to be effective in stimulating immune responses to protein antigens. Initial experiments were tested with the following parameters: the effect of one (primary, week 0) against two (primary and secondary, weeks 0 and 6) rounds of immunization (Example 1), kinetics of the antibody response (Example 2 ), dose of HBsAg liposome preparations (Example 3; 3 µg or 15 µg / mouse), route of administration (Example 4; intranasal or intramuscular), and physical form of the vaccine (Example 5; fresh or lyophilized). Samples were collected at the indicated times for the analysis of humoral and mucosal immune responses.

Negative controls included untreated mice, liposomes alone and HBsAg protein alone. Animals were also immunized with 3 µg of GSK intramuscular vaccine (Engerix ™ -B from GSK Biologicals, or "GSK HBsAg") as a positive control. Both genetically heterogeneous non-blood CD1 mice (N = 225 to 5 / group) and in inbred mice with a tendency to Th2 (N = 50 to 5 / group) were immunized for comparative purposes. CD1 mice are a non-consanguineous strain (that is, genetically heterogeneous) and are therefore representative of the diverse genetics found in humans.

For example, in a series of experiments, 10 mice were immunized at week 0 by the primary antigen. At week 6, a booster dose was administered to 5 of the 10 mice. Samples were collected (for example, from blood, feces and vaginal washes) at weeks 2, 4, 6, 8 and 12 for antibody determination by ELISA.

The examples below show representative data from these studies to demonstrate the optimal conditions for vaccination with the HBsAg liposomes in question. The humoral immune response to the different vaccines was measured by determining total serum antibody responses of total HBsAg using ELISA assays that were developed (previously). Although the samples taken at 2, 4, 6, 8 and 12 weeks after priming were analyzed, only the responses are shown at eight weeks after the first immunization for most of the examples.

Most of the charts include responses to the commercially available GSK HBsAg vaccine as a positive control, and serve as a benchmark that is to be achieved for test vaccines. No specific HBsAg responses were detected in any untreated mouse (data not shown). Closed symbols identify the responses of mice that received an immunization, while closed symbols with cross hatching (+) show the responses of mice that received both a primary immunization and a secondary immunization. The results are presented as the dilution of the sample tested (x-axis) versus the optical density at 450 nm in ELISA assays for HBsAg-specific total serum antibodies (y-axis). ELISA results expressed as arbitrary units were read at 450 nm (DO450).

For clarity, most of the data presented here are averages of the combined samples of 510 mice per experiment (equal amounts of individual samples from the groups of mice). Figure 2 is representative of the degree of variation observed between CD1 mice and individual Balb / c mice of an experiment.

In Figures 2-8, it is shown that in all the conditions examined, with the exception of a 3 µg dose of HBsAg liposome administered intranasally (Figure 4), secondary immunization significantly improved the specific serum antibody responses of HBsAg for both HBsAg liposomes and for the GSK HBsAg control vaccine. Therefore, liposomes are a very effective adjuvant and an administration vehicle for the HBsAg antigen and antibody responses are substantially increased after a second secondary immunization of identical reinforcement.

EXAMPLE 2: Kinetics of Antibody Responses in CD1 Mice after Intranasal Immunization (IN) with HBsAg encapsulated in liposomes (HBsAg liposomes)

Figure 3 illustrates serum titration of HBsAg specific antibodies after immunization of CD1 mice intranasally with 15 µg of HBsAg liposome. Significant reinforcement was observed in the titration of a specific HBsAg antibody if a second booster administration of the same Ag preparation was used. However, without a booster administration the titration reached its peak at 4 weeks after immunization. initial, and remained at similar levels until week 8. In addition, 15 µg of HBsAg liposome are at least as effective as the commercially available GSK vaccine at dosages of 3 µg.

EXAMPLE 3: Liposome Dose Response of HBsAg

CD1 mice were immunized, with or without a second booster administration, with 3 µg or 15 µg of HBsAg encapsulated in the liposome preparation. Figure 4 shows that 15 µg of HBsAg liposomes were at least as effective as the commercially available GSK vaccine (3 µg). However, 3 µg of HBsAg in the same liposome preparation was significantly less effective, and a second administration of reinforcement does not appear to result in a significant increase in titration.

EXAMPLE 4: Routes of Liposome Administration of HBsAg

To test the effect of the routes of administration, CD1 mice and Balb / c mice were immunized with the same HBsAg liposome preparation described above, through intranasal (IN) or intramuscular (IM) administration. Antibody titers were measured 8 weeks after (initial) immunization. In both CD1 and Balb / c mice, both by IN and IM administration of 15 µg of HBsAg liposomes, with or without a second booster, were at least as effective as 3 µg of the GSK vaccine (Figures 5 and 6) .

EXAMPLE 5: Effects of Fresh or Lyophilized Preparations of HBsAg Liposomes on Antibody Responses

To test whether the HBsAg liposome preparation has to be used fresh or lyophilized, CD1 mice were immunized with fresh or lyophilized liposome preparations, via intranasal or intramuscular administration. All serum Ig titers were measured 8 weeks after initial immunization.

Intranasal administration of HBsAg liposome was at least as effective as 3 µg of the GSK control vaccine if the liposome preparation was fresh (Figure 7). Comparable levels of titres were observed between the GSK vaccine and the fresh preparation of HBsAg liposomes, with or without reinforcement. Lyophilized HBsAg liposomes were slightly less effective than the fresh preparation in this experiment. However, in another separate experiment, the preparation of lyophilized HBsAg liposomes was slightly better than the fresh counterpart (data not shown).

A similar result was obtained with the IM administration (Figure 8). Both the preparation of fresh and lyophilized HBsAg liposomes had similar final titres compared to each other, and for the positive control of GSK.

These results indicate that reconstituted lyophilized preparations of HBsAg liposomes were generally as effective as fresh preparations when administered by IN or IM route. The effectiveness of lyophilized preparations will simplify the distribution and storage of future vaccine formulations.

EXAMPLE 6: The Effect of Liposome Size on Liposome Antibody Responses of HBsAg

The effect of liposome size was evaluated by encapsulating HBsAg (15 µg / mouse) in liposomes with a size of 4 µm, 1 µm and 0.2 µm. Quantitative ELISA assays were used to measure serum IgG levels specific for HBsAg after intranasal immunization with liposomes of a single size or after intranasal immunization with an equal mixture of all three sizes. It is possible that a mixture of liposomes of different sizes may be more immunogenic than a single liposome size. The total amounts of HBsAg and lipids were the same in the four groups.

Figure 9 shows that liposomes of size 1 µm and 4 µm were the most effective, while liposomes smaller than 0.2 µm are less effective in the generation of IgG in HBsAg specific serum. A mixture of equal parts of the three sizes generated an additive, but not a synergistic response. The predicted response, if the effects of the three sizes of the combined liposomes are additive, is shown in Figure 9 in the form of a vertical bar.

EXAMPLE 7: The Effect of the Liposome Proportion on Protein on Antibody Responses to HBsAg Liposomes

The importance of liposome ratios with respect to protein in the generation of serum IgG antibodies specific for HBsAg was determined by immunization of intranasally mice with HBsAg liposomes (15 µg / mouse, primed at week 0 and reinforced at week 6) in the conventional liposome content, or 1/2 and 1/3 of the liposome concentration. The reduction in the proportion of liposomes with respect to HBsAg by 1/2 and 1/3 reduced serum IgG responses disproportionately to 35% and 2% of the response obtained from conventional liposome preparation, respectively (n = 8 animals per group; serum IgG was measured in serum combinations of each group two weeks after reinforcement). Therefore, the amount of liposome used to encapsulate HBsAg has a significant effect for the generation of high levels of HBsAg-specific serum antibody responses.

EXAMPLE 8: Characterization of Th Polarized Responses Following Homologous Immunization Protocols

Cytokines are the hormonal messages responsible for most of the biological effects on the immune system, such as cell-mediated immunity and allergic responses. Although they are numerous, cytokines can be functionally divided into two groups: those that are pro-inflammatory and those that are basically anti-inflammatory, but that promote allergic responses.

T lymphocytes are a major source of cytokines. These cells have specific antigen receptors on their cell surface to allow recognition of foreign pathogens. They can also recognize normal tissues during episodes of autoimmune diseases. There are two subsets of T lymphocytes, which are distinguished by the presence of cell surface molecules known as CD4 and CD8. T lymphocytes expressing CD4 are also known as helper T lymphocytes, and these are considered the most prolific cytokine producers. This subset can be subdivided into Th1 and Th2, and the cytokines they produce are known as Th1 type cytokines and Th2 type cytokines.

Th1 type cytokines tend to produce pro-inflammatory responses responsible for the destruction of intracellular parasites and to perpetuate autoimmune responses. Interferon gamma (IFN-γ) and interleukin 12 (IL-12) are the main Th1 cytokines. CTL activity is also high in a Th1 type immune response. In contrast, Th2-type cytokines include interleukins 4, 5 and 13, which are associated with the promotion of IgE and eosinophil responses in atopy, and also interleukin 10 (IL-10), which has more of an anti-inflammatory response. The Th2 response provides support for maturation of B lymphocytes to immunoglobulin secreting cells, primarily activating the humoral defense mechanisms. CTL activity is generally low in a Th2 type immune response. In excess, Th2 responses also counteract the microbicidal action mediated by Th1.

In general, the optimal scenario seems to be that humans must produce a well-balanced response of Th1 and Th2, suitable for immune stimulation. However, in reality, the tendency of these responses to polarize is fundamental to the concept of the subset generation of Th1 and Th2. Therefore, a profile of

Th1 or Th2 cytokine production will often dominate during an immune response by preferentially amplifying a subset of Th and decreasing the opposite response. This polarized response seems to be fundamental for the defense of the host against many pathogenic organisms. Resistance to intracellular pathogens (such as viruses) often requires a predominantly Th1 response, while Th2 responses are typically necessary to combat extracellular parasites. Therefore, cytokines obtained from T lymphocytes produced by the host in response to an infectious agent determine the consequences of infection in many models of infectious diseases.

While a pro-inflammatory Th1 response is usually necessary to control intracellular infections, there is also a need to balance the response (Taylor-Robinson, Int J Parasitol. 28 (1): 135-48, 1998). It is important to produce a type 1 response powerful enough to keep the intracellular infection under control, while producing a type 2 or immunosuppressive response sufficient to prevent the protective response from causing damage to the host. Available data suggest that IFN-γ, IL-12 and IL-10 cooperate to keep the Th2 response in check. Therefore, a positive result after an infection requires an accurate assessment of the Th1 and Th2 responses, appropriate for the type of infection. This is not only in terms of quantity, but also where, when and for how long these responses occur.

Several factors have been proposed, including the properties of the antigens, the dose of antigen, place of exposure and the ongoing immune response in the host, to drive a T-lymphocyte response to a predominantly Th1 or Th2 phenotype. The responses of helper T lymphocytes to antigens can be characterized as polarized or mixed. The responses of polarized Th lymphocytes are obtained from a population trend of antigen-specific Th lymphocytes to a Th1 or Th2 cytokine profile, and are reflected by the proportions of IgG1: IgG2a specific for antigens of <0.5 and> 2.0, respectively. The mixed Th lymphocyte responses may contain both populations of T lymphocytes, and are reflected by the specific IgG1: IgG2a ratios of antigens between 0.5 and 2.0. See Table B below.

Table B. Characteristics of Th1 and Th2 Polarized Responses

Immune response

Type 1 (Th1) Type 2 (Th2)

Humoral Immunity

Proportion of IgG1: IgG2a <0.5 IgG1 ratio: IgG2a> 2.0

Cytokine Secretion

↑ IFN-γ, IL-12; ↓ IL-4, IL-10 ↑ IL-4, IL-10; ↓ IFN-γ,

CTL activity

high Low

The nature of the immune responses to HBsAg vaccine formulations were evaluated by determining the ratios of IgG1: IgG2a in the serum of CD1 and Balb / c mice 8 weeks after primary immunization. Quantitative ELISA assays were designed to measure IgG1 and IgG2a antibodies specific for HBsAg. Figures 10 and 11 show the proportions of IgG1: IgG2a in the serum of CD1 (Figure 10) and Balb / c mice (Figure 11) immunized by a commercially available GSK HBsAg vaccine, a DNA HBsAg vaccine and a HBsAg liposome vaccine administered intranasally or intramuscularly. Two vertical lines at 0.5 and 2.0 at each panel demarcate three different antibody response patterns (Th1, mixed or neutral, or Th2) in the regions from left to right.

According to the published results, the commercially available HBsAg vaccine preferably generated a Th2 type immune response, both in strains of mongrel CD1 mice and in strains of inbred Balb / c mice. As expected, the HBsAg DNA vaccine generated a Th1 type immune response in both strains. The administration of intranasal HBsAg liposomes generated a mixed response in CD1 mice and a Th2 response in Balb / c mice. In contrast, administration of intramuscular HBsAg liposomes generated a neutral immune response in both strains of mice.

The GSK vaccine was the human alum-based formulation designed for intramuscular administration (Engerix ™ -B of GSK Biologicals, adw subtype). The HBsAg DNA vaccine was a plasmid DNA (pRc / CMV-HBs (S) and was purchased from a commercial source (Aldevron, Fargo, ND). The plasmid expresses the small hepatitis B surface antigen under the control of the promoter CMV immediate-early. (Reference: Davis, HL,

M. L. Michel and R. G. Whalen. DNA-based immunization for Hepatitis B induces continuous secretion of antigen and high levels of circulating antibody. Human Molecular Genetics 2: 1847-1851. 1993).

EXAMPLE 9: Unique IgA Responses After Intranasal Administration of HBsAg Liposomes

From the late 1960s, mucosal immunity was recognized as an important defense against respiratory viruses when correlation with protection against respiratory viruses was shown in humans. At the same time, it was discovered that the IgA class of antibodies was especially prevalent in the respiratory tract and other mucous surfaces and that it was the mediator of mucosal immunity. Other studies have confirmed that the

Mucosal immunity could ideally be stimulated by developing protection against infection through vaccination at the local level.

Both IgG and IgA antibodies appear to be quite strain specific. With natural infection by respiratory viruses, such as the influenza virus, both a humoral immune response and a systemic immune response can occur. The level of anti-viral antibodies in the serum also correlates with protection.

One aspect of the present invention provides a method for eliciting mucosal immune responses by administering HBsAg in liposomes directly to the mucosal sites. An IgA sandwich-specific ELISA assay was designed to measure the specific IgA of HBsAg in serum and mucosal sites. Serum, feces and vaginal washes were tested from all experimental groups that have been previously analyzed and some saliva samples for IgA. The titration criteria were established for each sample combination (N = 4-5 mice per group) by serial dilution of test samples. Titration titration criteria were defined as an OD at 450 nm in the IgA ELISA assay of more than twice the value observed in the samples of mice of the same untreated strain not immunized in the same sample dilution.

The only samples that showed detectable IgA responses were from the mice that received the HBsAg liposome vaccine intranasally. Table C summarizes the results of the samples that tested positive for the HBsAg-specific IgA ELISA. IgA responses were detected in serum and in most of the samples obtained from mucosal sites after only one immunization. Booster with secondary immunization further increased IgA levels in most samples. Additionally, local intranasal immunization generated secretory IgA responses at all sampled remote mucosal sites, including faecal granules, vaginal washes and saliva (data not shown for saliva, due to the limited number of samples). These results demonstrate that immunization with liposomes of HBsAg by an (intranasal) route generated immunity of mucosal IgA specific to HBsAg in all sampled mucosal sites. The HBsAg liposome vaccine, unlike HBsAg protein or liposomes alone, boosts serum immune Ig titers and also induces both serum IgA and secretory responses when administered intranasally.

None of the other immunogens produce detectable IgA responses (results not shown).

Table C: Summary of IgA responses in mice immunized with HBsAg liposomes

Immunization

Strain Serum IgA Fecal IgA Vaginal IgA Salivary IgA

HBsAg IN liposome (1 x)

CD1 Means, medium Negative ND§ NA¶

HBsAg IN liposome (2 x)

CD1 Means, medium Low ND§ Tall

HBsAg IN liposome (1 x)

Balb / c Low Low Means, medium NA¶

HBsAg IN liposome (2 x)

Balb / c Tall Tall Tall Tall

Serum samples, feces and vaginal washes were collected from five individual mice per group. Samples were processed individually, and sample combinations were made using equivalent volumes of sample per mouse. The titration criteria were determined for each sample and defined as a serum titre> 2 x that was observed for mice immunized without treating CD1 or Balb / c. The range of titration values that was used for the determination of the relative titration is shown below. § Undetermined. ¶ Sample not available.

Relative rating

Serum Stool Vaginal wash

Negative

<100 <100 <25

Low

100-200 100-200 25-100

Half

400-800 400-800 200-400

high

> 1600 > 1600 400-1600

EXAMPLE 10: Antibodies Responses to DNA Immunization Only from HBsAg are Weak

Recently, immunization of the DNA vaccine to generate a protective immune response in a host, including humans, has been described. See U.S. Patent No. 6,632,663 and WO 03/075955 A1 (incorporated herein by reference).

To test the efficacy of DNA HBsAg as an immunogen, CD1 mice and Balb / c mice were immunized intramuscularly with DNA HBsAg, with or without a second booster. The Ig in HBsAg specific total serum was measured 8 weeks after immunization as described above. The GSK HBsAg vaccine was used as a positive control.

Figure 12 indicates that, unlike HBsAg protein vaccines with protein adjuvants, intramuscular immunization with HBsAg DNA produced a weak antibody response to HBsAg in both strains of CD1 and Balb / c mice. The immune response elicited, even with a booster, is weak to moderate at best compared to the GSK unbalanced control vaccine.

EXAMPLE 11: Homologous Immunization of HBsAg from DNA Generates a Powerful CTL Activity and a Th1 Polarized Cytokine Profile

The immunization of HBsAg from DNA is a weak inducer of antibody responses (Example 10), but it is a very strong inducer of T-lymphocyte responses after immunization with HBsAg from DNA and intramuscularly (100 µg in the weeks 0 and 6, with a booster dose at week 11 to mobilize memory CTL), an average specific destruction of HBsAg of 87% ± 8% HBsAg was observed using the CTL assay in vivo as a reading (n = 4 mice ). Immunization with HBsAg from DNA also strongly polarized the cytokine response by HBsAg-specific T lymphocytes towards a Type 1 cytokine profile. Figure 13 shows the results of ELISPOT assays for IFN-γ and IL-10 by cells splenic mice immunized with HBsAg DNA. Both the HBsAg protein, which contains epitopes of T lymphocytes, and the immunodominant peptide CTL of HBsAg (Ishikawa, T. et al. 1998. J. Immunol. 161: 5842) generated high frequencies of IFN-γ secretory cells, but a very small number of IL-10 secretory cells. Therefore, homologous immunization with HBsAg of DNA generates a typically strongly polarized profile of Th1 type, in accordance with the proportions of IgG1: IgG2a of Th1 type that were observed in two different strains of mice (Figures 10 and 11).

EXAMPLE 12: Effects of Heterologous Immunization with HBsAg of DNA as a Primer and Liposomes of HBsAg as a Booster in Systemic and Mucosal Antibody Responses

Although intramuscular immunization of DNA with HBsAg liposomes was a weak stimulus for total antibody production (Example 11), it was very effective in stimulating immune responses mediated by Th1 polarized lymphocytes (Example 11). In order to determine whether DNA immunization was effective in generating memory T and B lymphocyte responses, a heterologous immunization protocol was developed. In this protocol, immunization with HBsAg of DNA was used as the primary dose, and liposomes of HBsAg were used as the secondary dose. If the DNA is effective in generating memory T and B lymphocyte responses, then these may reveal after reinforcement with a different form of the antigen (HBsAg liposomes). Thus, the invention provides an experimental design of heterologous immunization. Variations of the protocol are simply routine and are within the scope of the invention (see Example 16 for permutations in the protocol).

In a representative protocol, mice were first immunized and primed with a DNA HBsAg vaccine intramuscularly, and then exposed to a low dose of liposomes of HBsAg intranasally. In certain embodiments, the low dose is itself insufficient to elicit a significant immune response when administered alone. Compared to a dose sufficient to induce a large-scale immune response, the low dose is at most 50%, 40%, 30%, 20%, 10%, 5% or less.

Balb / c mice were immunized intramuscularly by 100 µg of DNA HBsAg at week 0, and again at week 6. At week 16, a booster of HBsAg liposome was administered intramucosally at a reduced dose of 3 µg. Serum samples were obtained 2, 4 and 6 weeks after reinforcement. As a positive control, CD1 mice were immunized intranasally with 15 µg of HBsAg liposomes with a second booster. Positive control serum was obtained 8 weeks after initial immunization. As a negative control, sera from untreated CD1 mice were obtained at week 8.

Figure 14 shows the results of this heterologous immunization protocol. Two rounds of immunizations with HBsAg of IM DNA or one round of single immunization of liposomes of HBsAg IN (low dose) induced weak serum antibody responses. However, by adding a second low dose booster with HBsAg liposomes (3 µg) IN to DNA immunization, the regimen markedly increased the response of total HBsAg specific antibodies to an even higher level than that observed in mice afterwards. of two rounds of HBsAg IN liposomes (high dose).

Quantitative ELISA assays were used to determine the amount of micrograms of HBsAg specific IgG in the serum of this experiment. IgG levels after two rounds of DNA were an average of 43.2 µg / ml, and increased to 248 µg / ml after boosting with HBsAg liposomes. Administration of the liposome preparation alone generated only 0.6 µg / ml of serum IgG.

It should be noted that immunization with DNA also strengthens the immune system of mucosal IgA responses, which are also mobilized after intranasal administration of a low dose of HBsAg liposome booster. Figure 15 shows that DNA and liposome components of the heterologous immunization regime alone do not stimulate detectable IgA responses. However, substantial IgA responses were induced in the serum and vagina using the heterologous DNA priming protocol and HBsAg liposome booster. Secretory IgA was also induced in stool samples using this protocol (data not shown). Figure 15 also shows typical serum IgA responses after priming and boosting with a higher dose of only HBsAg liposomes (15 µg of HBsAg), which are a benchmark for strong IgA responses. HBsAg liposome booster must be administered intranasally to generate serum or mucosal IgA responses. If the booster is administered IM instead of IN, only a low level of secretory IgA is generated despite the fact that circulating serum IgG responses are strong (data not shown).

IgG antibodies specific for HBsAg were also detected in the lung and vaginal washings of mice to which the heterologous immunization protocol was administered (Figure 16). Neither untreated mice, nor mice that received HBsAg from IM DNA followed by white IN liposomes, generated detectable levels of IgG in the lung or vaginal washings that were above the background values. However, mice that were immunized with a combination of IM DNA HBsAg followed by HBsAg IN liposomes produced easily detectable mucosal IgGs in both the lung and vaginal sites. This is relevant since it has also been shown that mucosal IgG plays a protective role in the protection against certain pathogens, such as HSV-2 and influenza (Parr, EL et al. 1997. J. Virology 71: 8109- 8115 for HSV-2; Tamura, S. and T. Kurata. 2004. Jpn. J. Infect. Dis. 57 (6): 236-47; Renegar, KB et al. 2004. J. Inmunol. 173 (3 ): 1978-86).

The Applicants also determined the proportion of IgG1: IgG2a to evaluate the effect of IN reinforcement with HBsAg liposomes on the polarization of the T lymphocyte response, following the heterologous immunization protocol. Figure 17 shows the proportions of IgG1: IgG2a in the sera of individual mice after boosting with HBsAg liposomes. Significantly, the addition of this booster in the DNA immunization protocol did not change the immune responses towards a Th2 type profile. Antibody responses 4 weeks after the second DNA immunization showed a Th1 type trend (IgG1: IgG2a ratio of 0.47). A response with a tendency to Th1 was still present after intranasal reinforcement with liposomes of HBsAg (IgG1: IgG2a ratio of 0.50). This experiment shows that immunization with DNA establishes a solid and stable base of Th1 type memory that is mobilized efficiently when HBsAg liposomes are administered intranasally.

The heterologous immunization protocols in question promote Th neutral or Th polarized type 1 responses and cell-mediated immunity, while mucosal IgA and IgG responses are optionally induced (if the booster dose antigen-liposome preparation is administered intranasally). For protective immunity against intracellular pathogens, it is often desirable to initiate cell-mediated immune responses, particularly to improve Th1 cytokine secretion and for the generation of CLT. This is particularly useful for certain embodiments, in which it may be desirable for an antigen and / or immunization protocol to result in a Th1 response, rather than a Th2 response or a mixed type response, since the responses of Th2 may be associated with certain undesirable side effects similar to an allergic reaction, especially in children.

In other embodiments, in which the initial dose is the preparation of liposomes of HBsAg in question administered intranasally, and the booster dose is HBsAg of DNA administered intramuscularly, the resulting moderate immune response in the host animal tends to be of a mixed type, instead of a response of type Th1. This illustrates the argument that the order of the heterologous immunization in question is important to achieve the Th1 response.

EXAMPLE 13: Effects of Heterologous Immunization with DNA HBsAg as a Primer and Liposomes of HBsAg as Reinforcement on the Avidity of Serum IgG Antibodies for HBsAg

The avidity of the antibodies (that is, the total strength of the antigen-antibody binding) can play an important role in the control of pathogens, with the highest avidity antibodies generally being more effective than the low avidity antibodies. To determine the binding strength of serum IgG antibodies specific for HBsAg for the HBsAg antigen, a quantitative avidity test for IgG was developed using the chaotropic urea reagent to disrupt antigen-antibody complexes. Antibodies with greater avidity require higher concentrations of urea to dissociate the antigen bound to the antibodies. Figure 18 shows representative results of the avidity of IgG in mice immunized by a homologous immunization regimen (left panel) and a heterologous immunization regime (right panel). The results are represented as the avidity indices, which are µg of free IgG in the absence of urea divided by µg of free IgG in the presence of urea. The maximum ratio that can be obtained in this test is 1.0, which is shown as a dashed line at the top of each graph.

Homologous immunization with HBsAg liposomes creates avidities that are very similar to those obtained with the current GSK alumina vaccine of HBsAg used in humans. Immunization with HBsAg from DNA also generates good avidity responses. Heterologous immunization with HBsAg of DNA followed by liposomes of HBsAg increases avidity antibodies further, exceeding the reference response of the commercially available human vaccine. Administration of the liposome booster of HBsAg intranasally results in the most avid antibodies; However, IM administration is also very effective. Higher avidity antibodies are expected to be more effective in antigen neutralization, demonstrating the superiority of the heterologous immunization protocol over homologous immunization regimens with the commercially available GSK vaccine (IM) or HBsAg liposomes ( IN or IM).

EXAMPLE 14: Heterologous Immunization with DNA HBsAg as a Primer and Liposomes of HBsAg as Reinforcement Overcomes the Lack of Sensitivity to HBsAg in C57BL / 6 Mice

C57BL / 6 mice are not sensitive to the HBsAg protein (ref .: Schirmbeck, R. et al. J. Virol. 69 (10): 5929-34), since approximately 5-10% of human beings are who receive the commercially available GSK vaccine for HBsAg. To determine if heterologous immunization can induce immune responses in mice that do not respond, the following experiment was performed (Table D). A group (Group A) of C67BL / 6 mice was immunized with two rounds of HBsAg liposomes administered intranasally. As expected, these mice could not generate a detectable serum IgG level of anti-HBsAg. Then, these mice and a second group of untreated mice (Group B) were immunized with two rounds of HBsAg of DNA administered intramuscularly. Serum IgG levels in both groups of mice in response to the DNA vaccine were comparable (12.8 µg / ml and 8.0 µg / ml, for Groups A and B, respectively). Then, each group was further potentiated with HBsAg liposome before the determination of final serum IgG levels. Surprisingly, both groups of C57BL / 6 mice demonstrated high equivalent levels of serum IgG of anti-HBsAg (Group A, 238 µg / ml; Group B, 310 µg / ml). The results of this experiment illustrate three points: 1) heterologous immunization can stimulate high antibody responses in non-sensitive mouse strains; 2) the order of immunization is essential for the induction of serum IgG responses; 3) synergistic antibody responses are observed after heterologous immunization.

Table D. Immunization of HBsAg from DNA followed by liposomes of HBsAg IN overcomes the lack of

HBsAg sensitivity in C57BL / 6 mice

Treatment Groups

A: Serum IgG (µm / ml) B: Serum IgG (µm / ml)

A. 2 x HBsAg IN liposomes

0 ---

B. 2 x HBsAg of IM DNA

12.8 8.0

C. HBsAg IN liposomes

238 310

EXAMPLE 15: Heterologous Immunization Generates CTL Activity in vivo and Cytokine Secretion with Th1 Trend

The protective immunity of many intracellular pathogens often requires a Th1-prone immune response with high IFN-γ production, and the generation of cytolytic T lymphocytes (CTLs) that can destroy pathogen-infected cells. To evaluate the cell-mediated immune responses generated by vaccine administration protocols, Applicants have established in vivo assays to measure CTL responses, and ELISPOT assays to enumerate the frequencies of HBsAg-specific IFN-γ and IL10 secretory cells. (Th1 and Th2 responses, respectively).

CTL assays in vivo have recently been published in the literature. Unlike in vitro CTL assays, they provide a real measure of the ability of CTLs to destroy targets in the in vivo environment. Applicants measured CTL activity in vivo in mice that received both rounds of HBsAg of IM DNA followed by a low dose of liposome booster HBsAg IN. After the collection of samples for the measurement of antibody responses (six weeks or more after the IN booster) was completed, the mice received a DNA booster to mobilize memory T lymphocytes in the activated effector cells. 7 days later, the activity of the mobilized CTL effector cells was quantified by determining their ability to specifically destroy dye-labeled targets pulsed with HBsAg peptides that were adopted adoptively intravenously to the mice, as described in the Procedures. The disappearance of the CFSEalto population marked with peptide pulsed dye with respect to the CFSE population marked with unpressed dye is the measure of specific peptide destruction. The percentages of these two populations in the untreated receptor cohorts serve as an internal control for cell grafting. The percentage of CFSEbajo and CFSEalto donor cells in the spleens was determined by flow cytometry analysis.

Representative flow cytometry analyzes are shown in Figure 19 for an assay. The left panel shows CFSEbajo (R2 in the graph) and CFSEalto (R3 in the graph) populations of donor cells that were collected from the spleens of untreated mice 22 hours after injection of CFSE-labeled target cells. CFSE fluorescence is shown in the FITC or FL1 channel on the x axis. FL1 fluorescence is shown against irrelevant FL2 fluorescence only in order to exclude autofluorescent cells from the analysis. The right panel shows the same analysis of mice that had received the heterologous immunization protocol. The disappearance of a high CFSE population (pulsed with specific peptides) from the R3 region indicates specific destruction by CTL.

High levels of specific CTL destructive activity of HBsAg peptides were observed in Balb / c mice that were primed intramuscularly with DNA HBsAg and enhanced intranasally with HBsAg liposomes. CTL activity in vivo was an average of 78% (specific destruction range of 65-91%) in a cluster of four mice.

Spleen cells from mice that received the heterologous immunization regimen were also cultured in vitro for the measurement of IFN-γ and IL-4 secretory cells by ELISPOT assays. ELISPOT assays were established using commercially available antibody pairs and reagents (BD Pharmingen) and Multiscreen-IP multi-well ELISPOT plates (Millipore, Hopkington, MA). In vitro cultures were stimulated with HBsAg peptide at a concentration of 10 µg per 2.5 x 106 cells per ml for 20-22 hours. Peptide-free cultures were used as a negative control, and cultures stimulated with anti-CD3 or Con A were used as a positive control. The points were analyzed quantitatively by CTL Inc.

Applicants observed high frequencies of HBsAg-specific IFN-γ secreting cells, but only very low frequencies of HBsAg-specific IL-10 secreting cells in mice immunized with the heterologous immunization protocol (data not shown). These results directly confirm the conclusion derived from the analysis of serum IgG1: IgG2a ratios, that the heterologous immunization protocol generates an immune response with a tendency to Th1 type cytokines.

EXAMPLE 16: Variations in the Administration Protocol for Components with DNA HBsAg and HBsAg Liposomes of the Heterologous Immunization Protocol

Other studies determined the extent to which the two parameters of the heterologous immunization protocol, DNA priming and antigen-liposome reinforcement, could vary while retaining good immunogenicity for CTL and / or antibody responses. Table E shows the results of CTL destructive activity in vivo and serum IgG responses in mice in which the primary dose of DNA varied with respect to dose and time of administration. The first protocol (A) shown consists of administration of DNA at weeks 0 and 3 (total dose of 200 µg) and reinforcement of liposomes of HBsAg at week 6. This protocol generates This protocol generates almost the maximum destructive activity of specific CTL and high levels of IgG in HBsAg specific serum, as shown in the previous examples. The reduction of DNA from 2 administrations for a total of 200 µg of an administration for a total of 100 µg (B) produces the same levels of CTL responses and antibodies. A further modification consisting in dividing the primary dose of 1 x DNA into two parts administered over a period of three days, and the shortening of the secondary administration of liposomes by three weeks (C) conserved the high destructive activity of CTL and The antibody response. In a primary dose of reduced DNA

(D) there is an indication that both CTL levels and antibody levels are beginning to decrease. In DNA (E) less than 100 times, CTL activity is still approximately 2/3 of the maximum, while antibody levels are almost undetectable. Therefore, significant variations are possible both in the dose and at the time of booster immunization with DNA while retaining the desired result of high levels of CTL and antibodies.

Specific CTL of HBsAg in vivo and serum IgG in Balb / c mice

Table E. Effect of the different priming regimes with DNA on the generation of destructive activity

Treatment groups

% of destruction in vivo Serum µg / ml

A. 2 x DNA at weeks 0, 3 (200 µg total) + HBsAg IN liposomes at week 6

87 ± 8 176

B. 1 x DNA in 3 days (100 µg in total) + HBsAg IN liposomes at week 6

91 ± 4 197

C. 2 x DNA in 3 days (100 µg total) + HBsAg IN liposomes in week 3

90 ± 5 318

D. 2 x DNA in 3 days (10 HBsAg-lipo IN week 3) + HBsAg IN liposomes in week 3

76 ± 14 125

E. 2 x DNA in 3 days (1 HBsAg-lipo IN week 3) + liposomes of HBsAg IN in week 3

61 + 22 0.5

Legend of Table E. Groups of 5 mice were immunized as indicated. Serum combinations were collected two weeks after the last immunization for HBsAg specific serum IgG determinations. CTL assays were performed in vivo in individual mice 7 days after administration of a 1 x DNA (100 µg) IM booster as a stimulus to help reveal the different DNA priming. The numbers in parentheses in the treatment groups indicate the dose of total DNA administered by mouse.

The second variable examined was the volume of HBsAg liposome administered intranasally to mice that had been primed with DNA HBsAg. This was achieved by mixing HBsAg and empty liposomes, keeping the total amount of protein administered at 15 µg / dose and the liposome content constant. This protocol was

5 possible because it was shown that the mixture of HBsAg and liposomes provides responses that are approximately 70% of the responses observed when HBsAg is encapsulated in liposomes. Serum anti-HBsAg specific IgG levels were found to be within a similar range after instillation of 50, 40 or 30 µl inoculation volume.

10 Table F. Effect of HBsAg liposome volume on antibody responses using the

heterologous immunization protocol

Week 0 Immunization

Week 3 Immunization Week 5 Antibody Determination

Group¶

2 x 50 µg IM Total Volume for IN Immunization Serum IgG of anti-HBsAg µg // ml

TO

HBsAg DNA 50 µl (15 µg of HBsAg + liposome) 198

B

HBsAg DNA 40 µl (15 µg of HBsAg + liposome) 285

C

DNA DNA 30 µl (15 µg of HBsAg + liposome) 309

¶N = 8 mice per groups

EXAMPLE 17: Heterologous Immunization (primed with HBsAg DNA and HBsAg liposome booster) Generates Mucosal CTL and Provides Protection against Exposure of a Live Virus (VV-HBsAg)

15 To determine whether the heterologous immunization regimen generated local mucosal or systemic CTL, defined by its CD8 + IFNγ + phenotype, Balb / c mice were immunized with two rounds of IM DNA HBsAg followed by a round of HBsAg IN liposomes. Balb / c mice were immunized with two rounds of HBsAg of DNA followed by a round of empty liposomes, and non-immunized mice served as controls. The mice were stimulated

20 intranasally with the recombinant vaccinia virus expressing HBsAg (VV-HBsAg) 14 days after immunization with liposomes. The lung mononuclear cells (local mucosal compartment) and splenic cells (systemic compartment) were isolated and tested for the presence of CTL five days after exposure to the virus. Antigen-specific CTLs were identified by their CD8 + IFNγ + phenotype determined by flow cytometry analysis. Table G shows that untreated mice generated few CTLs in the lung or the

25 spleen Substantial numbers of CTL were generated in the lung of mice immunized with DNA and empty liposomes, and much smaller numbers were also generated in the spleen. However, mice immunized with a combination of DNA and HBsAg liposomes generated a massive expansion of HBsAg-specific CTLs locally in the lung, and a significant number in the splenic compartment. These data demonstrate that both elements of this heterologous immunization protocol generate large amounts of local CTL.

30 specific antigen in the lung mucosa after exposure to the virus. Significantly, antigen specific CTLs were also detected in the splenic compartment indicating a systemic response of antigen specific CTL.

Table G. Generation of CD8 + IFNγ + CTL after heterologous immunization

Treatment groups§

% of CD8 + IFNγ + in lungζ % of CD8 + IFNγ + in spleenζ

Untreated

0.9% 0.05 ± 0.05%

DNA HBsAg 2 x White liposome 1 x

38% 0.44 ± 0.11%

2 x DNA HBsAg 1 x HBsAg liposome

73% 1.79 ± 0.31%

§ Balb / c mice were immunized as indicated (treatment groups; n = 5 mice per group) at four week intervals. Mice were immunized with 100 µg of HBsAg of DNA intramuscularly (50 µl per quadriceps), and 50 µl of vacuum of liposomes of HBsAg intranasally. Mice were infected with 2 x 105 PFU of VV-HBsAg intranasally in a volume of 50 µl 14 days after the last immunization. ζ Immunofluorescence analysis was performed in pulmonary mononuclear cells (n = 1 per group) and in splenocytes (n = 5 per group) five days after exposure to the virus. The cells were stimulated in vitro for five hours with 10 µm of HBsAg CTL peptide per 106 cells. Then, the cells were stained for surface CD8 and intracellular IFN-γ.

Figure 20 shows the results from an exposure to VV-HBsAg administered intranasally to three cohorts of mice: untreated mice, mice that received HBsAg from DNA and white liposomes IN, and mice that received HBsAg from DNA and liposomes from HBsAg IN. Logarithmic PFU (virus plaque forming units) 5 per lung were determined five days after exposure to VV-HBsAg. Untreated virgin mice have high titers of viruses in the lung, with an average of 3.72 logarithmic PFU per lung (11,038 PFU). Mice that received single DNA and liposomes had two logarithms of less PFU per lung than untreated mice (1.86 logarithmic PFU or 156 PFU). Mice that received HBsAg DNA and liposomes had no detectable PFU of the virus in the lung, indicating complete clearance of the virus from the exposure site.

10 mucosal Since the HBsAg protein in this construct will only be expressed intracellularly, the clearance of the virus is probably due to CTL activity in the lung compartment.

IX. SUMMARY

15 Table H summarizes the characteristics of the immune responses that were generated with the HBsAg vaccine administration platforms. Eleven different immunization protocols (homologous and heterologous) were developed and immune responses to these protocols were characterized. The immunization schedule and vaccine components are as indicated. All antibody responses were determined two weeks after the last secondary immunization. It should be noted that protocols 8 and 9 are short versions of the

20 heterologous immunization platform. Using HBsAg as a model antigen, Applicants have demonstrated that immune responses can target specific results (i.e., antibody response levels, cytokine profiles of T helpers (Th1 vs. Th2), development of secretory IgA mucosal immunity , and provocation of cytolytic T lymphocytes). These studies provide a rational basis that can be used to adapt immune responses to generate protective immunity against specific pathogens in the

25 specific circumstances to favor immune responses of type Th1 or Th2, or a mixed / more balanced response.

Table H. Summary of the characterization of immune responses to HBsAg vaccines

Experimental design

Immunization (week 0) Immunization (week 3) Immunization (week 6) Antibody response Avidity of antibodies Th type Mucosal antibody responses

Homologous Immunization

one

Liposome, IM liposome, IM - high half Th2 none or low

2

Liposome, IN liposome, IN - half half Th2 good

3

DNA, IM DNA, IM - low half Th1 none or low

4

HBsAg vaccine from GSK, IM HBsAg vaccine from GSK, IM - high half Th2 none or low

Heterologous Immunization

5

DNA, IM DNA, IM liposome, IN high high Th1 good

6

DNA, IM DNA, IM liposome, IM high high Th1 none or low

7

DNA, IM DNA, IM mixture of liposome + antigen, IN high high Th1 good

8

DNA, IM liposome, IN - high high Th1 good

9

DNA, IM, dose divided into 3 days liposome, IN - high high Th1 good

10

DNA, IM + liposome, IN DNA, IM + liposome, IN - high ND Th2 good

eleven

Liposome, IN liposome, IN I DNA, IM medium, without synergy ND Th2 good

Abbreviations: Liposome: HBsAg encapsulated in liposome 1-4 µm in diameter DNA: HBsAg IM DNA: intramuscular injection; IN: intranasal administration

In summary, the results show that specific doses of HBsAg, when encapsulated in liposomes of a certain composition and size (such as 4 µm medium size), are potent vaccines for serum antibody production. Strong responses are observed when the vaccine is administered intranasally or intramuscularly. In contrast, intramuscularly injected HBsAg DNA is less effective in inducing a serum-specific HBsAg antibody. Applicants noted that mice tolerated intranasal vaccines in all formulations without evidence of distress or mortality.

Only intranasal immunization with HBsAg liposome generated systemic and mucosal IgA responses, while all other immunizing regimens failed to produce IgA response. And finally, the heterologous immunization regimen with HBsAg DNA vaccine via IM, followed by HBsAg liposome via IN is an ideal vaccine administration platform, since it produces synergistic serum antibody response, a strong mucosal IgA response and in serum, and a potentially enhanced antigen-specific T lymphocyte response and IFN-gamma secretion without changing the Th1 type response established by priming the DNA vaccine.

The biodistribution of HBsAg liposomes was not measured directly. However, the ability of vaccine formulations to stimulate local immunity of mucosal surfaces was determined and the results presented above.

Generally, the nomenclature used and the laboratory procedures used in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. These techniques are explained in detail in the bibliography. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989); Perbal, &quot; A Practical Guide to Molecular Cloning &quot;, John Wiley &amp; Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) &quot; Genome Analysis: A Laboratory Manual Series &quot;, Vol. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in United States Patents No. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; &quot; Cell Biology: A Laboratory Handbook &quot;, Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton &amp; Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), &quot; Selected Methods in Cellular Immunology &quot;, W. H. Freeman and Co., New York (1980); The available immunoassays are described in detail in the patent and scientific literature, see, for example, Pat. of the United States No. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); &quot; A Practical Guide to Molecular Cloning &quot; Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; &quot; PCR Protocols: A Guide To Methods And Applications &quot;, Academic Press, San Diego, Calif. (1990); Marshak et al., "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996); each of which is incorporated by reference as if they were fully exposed in this document. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader.

Claims (19)

1. A primary preparation for intramuscular administration comprising a nucleic acid encoding an antigen: and a secondary preparation for intranasal administration comprising the antigen encapsulated in liposomes, to elicit an immune response in an animal in which the administration of the secondary preparation produces an immune response that comprises:

(one)

an increase in the serum level of the antigen specific IgA compared with the serum level of the antigen specific IgA prior to administration of the secondary preparation and,

(2)

an increase compared to an untreated animal in the proportion of antigen-specific CD8-IFNγ + cytotoxic T lymphocytes in both the lung and spleen.

2.

The composition of claim 1, wherein the immune response elicited tends to a Th1 response in a host animal.

3.

The composition of claim 1 or claim 2, wherein the desired immune response is against a pathogen and the antigen is a pathogen specific antigen.

Four.

The composition of claim 1, wherein the liposomes in the secondary preparation have an average of about 0.5-5 µm in size.

5.

The composition of claim 3, wherein the immune response confers immunity against the pathogen.

6.

The composition of claim 5, wherein the immune response protects animals from exposure by said pathogen.

7.

The composition of one of claims 3-6, wherein the pathogen is a virus.

8.

The composition of claim 7, wherein the virus is HBV, and the pathogen specific antigen is HBsAg.

9.

The composition of claim 8, wherein the animal is a human being.

10.

The composition of claim 1, in the secondary preparation does not contain an adjuvant other than liposomes.

eleven.

A kit for eliciting an immune response in a host animal comprising: a) a first immunizing component for intramuscular administration comprising a nucleic acid encoding an antigen; b) a second immunizing component for intranasal administration comprising the antigen encapsulated in liposomes; c) an instruction for a user to administer to the animal the first immunizing component followed by the administration of the second immunizing component to elicit an immune response in the animal.

12.

The kit of claim 11, wherein the desired immune response is against a pathogen and the antigen is a pathogen specific antigen.

13.

The kit of claim 11, wherein the liposomes in the second immunizing component have an average of about 0.5-5 µm in size.

14.

The kit of claim 12 or claim 13, wherein the pathogen is a virus.

fifteen.

The kit of claim 14, wherein the virus is HBV, and the pathogen specific antigen is HBsAg.

16.

The kit of one of claims 11-15 in which the animal is a human being.

17.

The kit of claim 11, wherein the second immunizing component does not contain an adjuvant other than liposomes.

18.

The composition of claim 1, wherein the secondary preparation consists of an antigen encapsulated in liposomes in a suitable solvent.

19.

The kit of claim 11, wherein the second immunizing component consists of an antigen encapsulated in liposomes and a suitable solvent.